Multivalent constructs for therapeutic and diagnostic applications

ABSTRACT

The invention provides compositions and methods for therapeutic and diagnostic applications.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/440,201 filed on Jan. 15, 2003, and U.S. Provisional ApplicationSer. No. 60/360,821, filed on Mar. 1, 2002, both of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods for therapeutic anddiagnostic applications.

BACKGROUND OF THE INVENTION

Researchers have long been attempting to exploit the ability oftargeting moieties or ligands to bind to specific cells (via receptorsor otherwise) to target compositions such as detectable labels ortherapeutic agents to particular tissues of an animal (especially ahuman). In such situations, the ability of the targeting moiety to bindto the target (e.g., affinity, avidity, and/or specificity)significantly impacts the ability to successfully target the desiredtissues.

Numerous attempts have been made to use natural (e.g. polyclonal) andmonoclonal antibodies, as targeting moieties in vivo. However, use ofsuch antibodies present certain disadvantages, such as unacceptablelevels of antigenicity—even for humanized antibodies. In addition,natural antibodies are difficult to produce in recombinant form, due tothe number of chains, disulfide bonds, and glycosylation. Naturalantibodies also present pharmacokinetic problems. Antibodies posesignificant problems in imaging and radiotherapeutic applicationsbecause, due to their large size, accumulation in extravascular targettissue and clearance from the vascular system are both slow. Thisproblem is especially critical when dealing with solid tumors, whichpresent additional barriers to the ingress of large blood borncompounds. Similar problems occur with antibodies used for imaging usingother modalities, such as magnetic resonance imaging (MRI), ultrasoundand light. If the antibody is radiolabeled with a diagnostic ortherapeutic radionuclide, lower target to background ratios result inthe images. In addition, an undesirable distribution of radiationexposure between the tumor and normal tissues occurs.

In attempts to solve these problems, efforts have been directed towardsthe construction of smaller entities with similar binding affinitiesusing the essential features of the natural antibody binding regions.The building blocks are typically single-chain Fv fragments (scFv) whichare monovalent. Combining fragments of this type so that they have thebivalent or multivalent properties of the antibodies has beenproblematic. In order to dock to a surface it is an advantage that thetwo binding sites on the antibody are connected via a flexible hinge tothe constant region. Thus, in order to imitate the binding efficacy ofantibodies, not only must the binding site be recreated, but so alsomust the bivalency (or higher valency) and the flexibility. Thisflexibility is needed because the protein backbone that makes up thenonbinding region of the scFv is still bulky relative to the bindingsite. Once an appropriate method has been devised to join two scFvfragments together, different scFv fragments can be joined together aswell as more than the customary two scFv moieties present in naturalantibodies. Certain scFv fragments, depending both on the VH/VLinterface and the linker length, can spontaneously dimerize ormultimerize. These “diabodies” are smaller than the natural antibody anddo not have the immunological properties of the Fc portion (whichactivates complement and/or binds to Fc receptors), which they lack. Thetwo (or more) binding sites are rotated relative to each other, and thusthe antigen must be correctly positioned to accommodate thispresentation.

“Miniantibodies” have properties similar to those of diabodies, butrather than a short 5-20 amino acid linker they have a relatively moreflexible linker that allows freer orientation of the binding sitesrelative to each other, similar to in a natural antibody. Likediabodies, miniantibodies do not have the high molecular weight,immunologically active Fc dimer fragment. They can also be made bybacterial systems. Although they have desired advantages over naturalantibodies, miniantibodies still suffer from having a relatively largesize, which affects their pharmacokinetics, and must be made usingbiological methods. The smallest miniantibody is about 120 kDa in size.

Attempts have been made to use bispecific antibodies (e.g. antibodiesthat bind to two separate targets) to overcome one of the majordeficiencies of antibodies, namely, that the size of the antibodiesslows accumulation in the extravascular target tissue and clearance fromthe blood. The bispecific approach taken has been referred to as“pretargeting.” This approach uses a two-step protocol. A bispecificantibody with at least one arm that recognizes a tumor-associatedantigen and at least one other arm that recognizes an epitope on adiagnostic or therapy agent is given as a first injection. After theunbound antibody has substantially cleared non-target tissues and hasreached a maximum level in the tumor, the smaller, bispecificantibody-recognizable diagnostic or therapeutic agent is given. It ishoped that the latter agents distribute more rapidly throughout thebody, and either bind to the bispecific antibody localized at the tumor,or are cleared via the kidneys.

An alternative to this approach attempts to use a mixed antibodyavidin/biotin system in a two-step procedure. For example, a targetingantibody is conjugated with either avidin or biotin and then is injectedwhereupon it localizes in the tumor of interest. Thereafter, eitherbiotin or avidin (depending on which was coupled to the targetingantibody), bearing an imaging or radiotherapeutic radionuclide, isinjected and becomes localized at the site of the primary antibody bybinding to avidin or biotin respectively.

Another approach to the use of antibodies as targeting moieties forradiopharmaceuticals or other diagnostic imagining agents has attemptedto use a bivalent hapten to increase the avidity for the cell boundbispecific antibody over that of the circulating antibody. This approachrelies on bidentate binding occurring with the cell bound antibodies,because the surface density on the cells is sufficiently high, but notoccurring with the circulating antibodies, because the concentration istoo low. In effect, the system makes use of the increase in aviditycaused by the closer presentation of the antibodies/antigen on thecells.

Peptides have also been used as targeting moieties. In an attempt toimprove the binding bi-specific peptide constructs have been preparedwith two or more peptide based targeting agents selective for differenttargets. For example, a hybrid peptide having ligands to two targetsselected from the somatostatin-, GRP-, CCK-, Substance P-, or VIPreceptor and α_(v)β₃ integrin was reportedly made and tested for theability to bind to tumor cells. The initial evaluation showed noimproved tumor uptake for the multiple ligand systems investigated. Theinvestigators assumed that steric impairment leads to a reduction of thereceptor affinities of the dimeric structures. Others have tested anRGD-DTPA-Octreotate hybrid peptide targeted towards both the α_(v)β₃integrin and the somatostatin-2 receptor for the ability to increase thetumor uptake over that of a peptide selective for one or the othertargets. The different binding affinities of the two targeting moietiestowards their targets, blood vessels and tumor cells, respectively,resulted in the avidity for tumors being dominated by the stronger(somatostatin mediated) interaction.

A variation of these approaches uses a bispecific diabody targeted totwo different epitopes on the same antigen. This approach attempts toincrease the avidity of the construct for the target, because, althoughthe binding is monovalent for each epitope, the construct as a whole isbivalent to its target, as each of the binding epitopes is locatedwithin the same target molecule. In the case of the single moleculetarget, scFv fragments have been found to have insufficient affinity andan increase in avidity was required.

Two rationales underlie the approaches described above. The firstrationale uses two different targeting moieties to overcome some of thepharmacokinetic problems associated with the delivery of antibodies tosolid tumors. The second rationale uses two different targeting moietiesto increase the avidity of the construct for a given target, such as asingle molecule or a whole tumor. However, all of the approachesdescribed suffer from various drawbacks. Thus, there remains a need fordiagnostic and therapeutic agents with increased affinity and or avidityfor a target of interest. There also remains a need for diagnostic andtherapeutic agents that, when administered in vivo to a mammal, haveacceptable pharmacokinetic properties.

Angiogenesis, the formation of new blood vessels, occurs not only duringembryonic development and normal tissue growth and repair, but is alsoinvolved in the female reproductive cycle, establishment and maintenanceof pregnancy, and repair of wounds and fractures. In addition toangiogenesis that occurs in the normal individual, angiogenic events areinvolved in a number of pathological processes, notably tumor growth andmetastasis, and other conditions in which blood vessel proliferation isincreased, such as diabetic retinopathy, psoriasis and arthropathies.Angiogenesis is so important in the transition of a tumor fromhyperplastic to neoplastic growth, that inhibition of angiogenesis hasbecome an active cancer therapy research field.

Tumor-induced angiogenesis is thought to depend on the production ofpro-angiogenic growth factors by the tumor cells, which overcome otherforces that tend to keep existing vessels quiescent and stable. The bestcharacterized of these pro-angiogenic agents is vascular endothelialgrowth factor (VEGF) (Cohen et al., FASEB J., 13: 9-22 (1999)). VEGF isproduced naturally by a variety of cell types in response to hypoxia andsome other stimuli. Many tumors also produce large amounts of VEGF,and/or induce nearby stromal cells to make VEGF (Fukumura et al., Cell,94: 715-725 (1998)). VEGF, also referred to as VEGF-A, is synthesized asfive different splice isoforms of 121, 145, 165, 189, and 206 aminoacids. VEGF₁₂₁ and VEGF₁₆₅ are the main forms produced, particularly intumors (see, Cohen et al. 1999, supra). VEGF₁₂₁ lacks a basic domainencoded by exons 6 and 7 of the VEGF gene and does not bind to heparinor extracellular matrix, unlike VEGF₁₆₅.

VEGF family members act primarily by binding to receptor tyrosinekinases. In general, receptor tyrosine kinases are glycoproteins havingan extracellular domain capable of binding one or more specific growthfactors, a transmembrane domain (usually an alpha helix), ajuxtamembrane domain (where the receptor may be regulated, e.g., byphosphorylation), a tyrosine kinase domain (the catalytic component ofthe receptor), and a carboxy-terminal tail, which in many receptors isinvolved in recognition and binding of the substrates for the tyrosinekinase. There are three endothelial cell-specific receptor tyrosinekinases known to bind VEGF: VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), andVEGFR-3 (Flt4). Flt-1 and KDR have been identified as the primary highaffinity VEGF receptors. While Flt-1 has higher affinity for VEGF, KDRdisplays more abundant endothelial cell expression (Bikfalvi et al., J.Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominatethe angiogenic response and is therefore of greater therapeutic anddiagnostic interest (see, Cohen et al. 1999, supra). Expression of KDRis highly upregulated in angiogenic vessels, especially in tumors thatinduce a strong angiogenic response (Veikkola et al., Cancer Res., 60:203-212 (2000)). The critical role of KDR in angiogenesis is highlightedby the complete lack of vascular development in homozygous KDR knockoutmouse embryos (Folkman et al., Cancer Medicine, 5^(th) Edition (B. C.Decker Inc.; Ontario, Canada, 2000) pp. 132-152).

KDR (kinase domain region) is made up of 1336 amino acids in its matureform. The glycosylated form of KDR migrates on an SDS-PAGE gel with anapparent molecular weight of about 205 kDa. KDR contains sevenimmunoglobulin-like domains in its extracellular domain, of which thefirst three are the most important in VEGF binding (Cohen et al. 1999,supra). VEGF itself is a homodimer capable of binding to two KDRmolecules simultaneously. The result is that two KDR molecules becomedimerized upon binding and autophosphorylate, becoming much more active.The increased kinase activity in turn initiates a signaling pathway thatmediates the KDR-specific biological effects of VEGF.

Thus, not only is the VEGF binding activity of KDR in vivo critical toangiogenesis, but the ability to detect KDR upregulation on endothelialcells or to detect VEGF/KDR binding complexes would be extremelybeneficial in detecting or monitoring angiogenesis. Diagnosticapplications, such as detecting malignant tumor growth, and therapeuticapplications, such as targeting tumoricidal agents or angiogenesisinhibitors to the tumor site, would be particularly beneficial.

Hepatocyte growth factor (also known as scatter factor) is amulti-functional growth factor involved in various physiologicalprocesses such as embryogenesis, wound healing and angiogenesis. It hasbecome apparent that HGF, through interactions with its high affinityreceptor (cMet), is involved in tumor growth, invasion and metastasis.In fact, dysregulated cMet expression (for example, the overexpressionof cMet in neoplastic epithelium of colorectal adenomas and in othercarcinomas as compared to normal mucosa) and/or activity, as well ashyperactivity of the cMet receptor through an autocrine stimulatory loopwith HGF, has been demonstrated in a variety of tumor tissues andinduces oncogenic transformation of specific cell lines.

In general, HGF is produced by the stromal cells, which form part ofmany epithelial tumors; however, it is believed that the production ofHGF by tumor cells themselves comprises the main pathway leading to thehyperproliferation of specific tumors. HGF/cMet autocrine stimulatoryloops have been detected in gliomas, osteosarcomas, and mammary,prostate, breast, lung and other carcinomas.

Interrupting the HGF interaction with the cMet receptor slows tumorprogression in animal models. In addition to stimulating proliferationof certain cancer cells through activation of cMet, HGF also protectsagainst DNA-damaging agent-induced cytotoxicity in a variety of celllines susceptible to hyperproliferative phenotypes (e.g., breastcancer). Therefore, preventing HGF from binding to cMet could predisposecertain cancer cells to the cytotoxicity of certain drugs.

In addition to hyperproliferative disorders, cMet also has been linkedto angiogenesis. For example, stimulation of cMet leads to theproduction of vascular endothelial growth factor (VEGF), which, in turn,stimulates angiogenesis. Additionally, stimulation of cMet also has beenimplicated in promoting wound healing.

In addition to identifying the cMet receptor as a therapeutic target forhyperproliferative disorders, angiogenesis and wound healing, the largediscrepancy between expression levels of neoplastic and correspondingnormal tissues indicates that cMet is an attractive target for imagingapplications directed to hyperproliferative disorders.

SUMMARY OF THE INVENTION

The present invention features multivalent constructs which bind to atarget of interest, as well as various methods related to the use ofthese constructs. The present invention uses small targeting moietieswhich bind to different binding sites of the same target, allowing forimproved localization to the desired target, and providing an improvedmeans for detecting, imaging and/or treating the target site.

Preparation and use of multivalent (e.g., dimeric or multimeric)targeting constructs which include two or more targeting moieties, forexample binding polypeptides, specific for different binding sites ofthe same target are described herein. These targeting constructs may belinked or conjugated to a detectable label and/or a therapeutic agent(as defined herein) and used to deliver the detectable label and/ortherapeutic agent to the target of interest. Thus, in addition to thetargeting constructs themselves, the invention includes diagnosticimaging agents and therapeutic agents useful in diagnostic imaging andtreating various disease states. Furthermore, the invention includes useof the targeting constructs of the invention themselves to treatdisease.

In one aspect, the present invention features a compound having aplurality of binding moieties, wherein at least two binding moietieshave specificity for different binding sites on the same target. Inpreferred embodiments, the plurality of binding moieties includes apolypeptide. In other preferred embodiments, the targeting moieties areall binding polypeptides which bind to different sites on the desiredtarget. In certain preferred embodiments, the target is a protein, areceptor, or a receptor/ligand complex and the binding polypeptides bindto different epitopes on the protein, the receptor, or thereceptor/ligand complex. In one embodiment, the target is a receptorinvolved in angiogenesis, hyperproliferative disorders or wound healing.In another embodiment the target includes a family of receptors, suchas, for example, protein-tyrosine kinase receptors. In a particularlypreferred embodiment, the target is KDR or the KDR/VEGF complex, and thebinding moieties, particularly binding peptides, bind to differentepitopes on KDR or the KDR/VEGF complex.

In another preferred embodiment, the target is the hepatocyte growthfactor (HGF) receptor (cMet) or the HGF/cMet complex, and the bindingmoieties (particularly binding polypeptides) bind to different epitopeson cMet or the HGF/cMet complex.

In further preferred embodiments, the affinity constant of a compound ofthe invention for its target is greater than the affinity constant of aconstituent polypeptide for the target.

In another aspect, the compounds of the invention include a labellinggroup or a therapeutic agent. In certain embodiments, the compounds ofthe invention include a linker between a binding moiety and thelabelling group. For example, the linker may include a substituted alkylchain, an unsubstituted alkyl chain, a polyethylene glycol derivative,an amino acid spacer, a sugar, an aliphatic spacer, an aromatic spacer,a lipid molecule, or combination thereof. Preferred labelling groupsinclude a radionuclide, a paramagnetic metal ion, an ultrasound contrastagent, and/or a photolabel. For example, preferred paramagnetic metalions used in compounds of the invention include Mn²⁺, Cu²⁺, Fe²⁺, Co²⁺,Ni²⁺, Gd³⁺, Eu³⁺, Dy³⁺, Pr³⁺, Cr³⁺, Co³⁺, Fe³⁺, Ti³⁺, Tb³⁺, Nd³⁺, Sm³⁺,Ho³⁺, Er³⁺, Pa⁴⁺, and Eu²⁺.

Radionuclides are also preferred detectable labels and therapeuticagents. The choice of radionuclide will be determined based on thedesired therapeutic or diagnostic application. In a preferredembodiment, where the detectable label is a paramagnetic metal or aradionuclide, the compounds of the invention include a chelator orchelating group. Preferable chelators inlcude DTPA, DOTA, DO3A, EDTA,TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, or MECAM. Foruse as a PET agent, a peptide may be complexed with one of the variouspositron emitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As,^(94m)Tc, or ¹¹⁰In. The heteromultimeric constructs can also be labeledby halogenation using radionuclides, such as ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I,¹²³I, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclides for scintigraphy orradiotherapy include ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm,¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb. ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy,⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi,²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au.The choice of metal or halogen will be determined based on the desiredtherapeutic or diagnostic application. For example, for diagnosticpurposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,and ¹¹¹In. For therapeutic purposes, the preferred radionuclides include⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁷⁵Yb, ¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au. A most preferred chelator used incompounds of the invention is 1-substituted 4,7,10-tricarboxymethyl1,4,7,10 tetraazacyclododecane triacetic acid (DO3A). Preferably, aradioactive lanthanide, such as, for example, ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, ¹¹¹In,or ¹⁶⁶Ho is used with DOTA or DO3A in compounds of the invention.

Compounds of the invention include chelators having the followingstructure:

where X is CH₂ or O;Y is C₁-C₁₀ branched or unbranched alkyl, aryl, aryloxy, arylamino,arylaminoacyl, or aralkyl comprising C₁-C₁₀ branched or unbranched alkylgroups, C₁-C₁₀ branched or unbranched hydroxy or polyhydroxyalkyl groupsor polyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups; J is C(═O)—,OC(═O)—, SO₂—, NC(═O)—, NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—, ahomopolyamide or a heteropolyamine derived from synthetic or naturallyoccurring amino acids; and n is 1-100. Most preferably, the compoundsfurther include ^(99m)Tc, ¹⁸⁶Re, or ¹⁸⁸Re.

In one embodiment, compounds of the invention include a chelator havingthe following structure:

Most preferably, the compound further includes ^(99m)Tc, ¹⁸⁶Re, or¹⁸⁸Re.

In another embodiment, the chelator comprises a compound having thefollowing structure:

Most preferably, the compound further includes ^(99m)Tc.

In other embodiments, compounds of the invention include a chelatorhaving the following structure:

where R is an alkyl group, such as CH₃. Most preferably, the compoundfurther includes ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, ¹¹¹In, or ¹⁶⁶Ho.

In yet another embodiment, compounds of the invention include a chelatorhaving the following structure:

where R is an alkyl group, such as CH₃. Most preferably, the compoundfurther includes ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, ¹¹¹In, or ¹⁶⁶Ho.

In other embodiments, the compound of the invention includes a chelatorhaving the following structure:

Most preferably, the compound further includes ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, ¹¹¹In,or ¹⁶⁶Ho.

Preferred ultrasound contrast agents for use in compounds of theinvention include phospholipid stabilized microbubbles or microballoonscomprising a fluorinated gas.

One preferred embodiment of the invention includes compounds comprisingat least two binding moieties with specificity for different bindingsites on a target. Preferably the target is a single receptor orreceptor/ligand complex such as, for example, KDR or the KDR/VEGFcomplex or cMet of the cMet/VEGF complex. In further preferredembodiments, the binding moieties bind to different epitopes on thereceptor or receptor/ligand complex. In a particularly preferredembodiment the binding moieties include a polypeptide. In otherpreferred embodiments, a compound of the invention includes apolypeptide having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, or SEQ ID NO:29. The invention also provides acompound having one or more of the foregoing amino acid sequences thathave been modified to include one or more amino acid substitutions,amide bond substitutions, D-amino acid substitutions, glycosylated aminoacids, disulfide mimetic substitutions, amino acid translocations, orhas been modified to include a retroinverso peptide, a peptoid, aretro-inverso peptoid, and/or a synthetic peptide. In preferredembodiments, the compound of the invention comprises SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:26, and/or SEQ ID NO:27. In a more preferred embodiment suchcompounds further include a labelling group or therapeutic agent asdescribed herein.

In another aspect, the invention features diagnostic imaging methodsusing compounds of the invention that include a labelling group. Methodsof the invention include the steps of administering to a patient apharmaceutical preparation that includes a compound of the inventionhaving a labelling group, and imaging the compound after administrationto the patient. In preferred embodiments, the imaging step includesmagnetic resonance imaging, ultrasound imaging, optical imaging,sonoluminescence imaging, photoacoustic imaging, or nuclear imaging. Inthese methods, the administering step may include inhaling, transdermalabsorbing, intramuscular injecting, subcutaneous injecting, intravenousinjecting, intraperitoneally injecting, intraarterial injecting orparenteral administration.

In another aspect, the compounds of the invention serve as therapeuticagents themselves and/or include a therapeutic agent. In certainembodiments, the compounds of the invention include a linker between abinding moiety and the therapeutic agent. For example, the linker mayinclude a substituted alkyl chain, an unsubstituted alkyl chain, apolyethylene glycol derivative, an amino acid or peptide spacer, asugar, an aliphatic spacer, an aromatic spacer, a lipid molecule, orcombination thereof. Preferred therapeutic agents for use with compoundsof the invention include a bioactive agent, a cytotoxic agent, a drug, achemotherapeutic agent, or a radiotherapeutic agent.

In another aspect, the invention features a method of treating a diseaseby administering to a patient a pharmaceutical preparation including acompound of the invention. In one embodiment, where one or more bindingmoieties of the compound inhibits a biological process that contributesto a disease state, the compound may be administered to treat thatdisease state. For example, the binding moieties may inhibit thebiological process by preventing or diminishing the activity of thereceptor (e.g. by competition with the natural ligand for the receptor,by directly inhibiting the receptor activity whether or not the naturalligand is bound, or by a combination of the two). Thus, aheteromultimeric compound of the invention, may inhibit the activity of,for instance KDR or cMet, and thus inhibit angiogenesis and/orhyperproliferation and consequently the diseases these processescontribute to. Therefore, the invention features a method of treating adisease by administering to a patient a pharmaceutical preparationincluding a compound of the invention alone or attached or linked to aseparate therapeutic agent. In preferred embodiments, the inventionfeatures a method of treating a disease associated with angiogenesis orhyperproliferation. In a most preferred embodiment, the disease isneoplastic tumor growth.

The invention also features a method of screening for heteromultimericcompounds having improved binding affinity. This method includes thesteps of preparing a labeled heteromultimeric compound comprising aplurality of binding moieties, wherein at least two binding moietiesbind to different binding sites of a target; contacting the labeledheteromultimeric compound with a target; determining a binding strengthof the labeled heteromultimeric compound (for example, by determiningthe dissociation constant); and comparing the binding strength (e.g.,dissociation constant) of the labeled heteromultimeric compound with thebinding strength (e.g., dissociation constant) of one or more individualbinding moieties. In preferred embodiments of this method one of thebinding moieties includes a polypeptide. In another preferredembodiment, the target is KDR or KDR/VEGF complex. In a preferredembodiment, one of the polypeptides used in this method is SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.Preferably, the method includes the step of identifying a labeledheteromultimeric compound having a binding strength (for example, asmeasure by the dissociation constant) that is less than the bindingstrength of a constituent binding moiety.

In one preferred embodiment, the invention features dimeric ormultimeric targeting constructs which include two or more KDR orVEGF/KDR complex binding polypeptides which bind to different bindingsites of KDR or the VEGF/KDR complex. Such polypeptides are described indetail in U.S. Ser. No. 60/360,851 and U.S. Ser. No. 60/440,441, both ofwhich are incorporated by reference herein in their entirety, and incopending application U.S. Ser. No. ______, entitled “KDR and VEGF/KDRbinding peptides and their use in diagnosis and therapy,” filed on thesame date as the instant application and incorporated by referenceherein in its entirety. These constructs are referred to herein as“KDR—targeting constructs.” The KDR targeting constructs exhibitimproved binding to KDR (e.g. increased specificity and/or affinityand/or avidity) compared to monomeric KDR or VEGF/KDR complex bindingpolypeptides, and compared to dimeric or multimeric constructs of asingle KDR-binding polypeptide. These preferred compounds may be linkedor conjugated to a detectable moiety and used to target thesecompositions to KDR-expressing cells, permitting imaging ofKDR-expressing tissue.

In another preferred embodiment, the invention features dimeric ormultimeric targeting constructs which include two or more cMet orHGF/cMet complex binding polypeptides which bind to different bindingsites of cMet or the HGF/cMet complex. Such polypeptides are describedin detail in copending application U.S. Ser. No. ______, entitled“Peptides that specifically bind HGF receptor (cMet) and uses thereof,”filed on the same date as the instant application and incorporated byreference herein in its entirety. These constructs are referred toherein as “cMet—targeting constructs.” The cMet targeting constructsexhibit improved binding to cMet (e.g. increased specificity and/oraffinity and/or avidity) compared to monomeric cMet or HGF/cMet complexbinding polypeptides, and compared to dimeric or multimeric constructsof a single cMet-binding polypeptide.

The cMet and KDR targeting constructs of the invention may be linked orconjugated to a therapeutic agent and used to localize the therapeuticagent to cMet- or KDR-expressing tissue. Alternatively or additionally,the cMet or KDR targeting constructs of the invention may also be usedas therapeutics themselves, as described herein.

In particularly preferred embodiments, the KDR targeting constructs ofthe invention include two or more of the following KDR and VEGF/KDRcomplex-binding polypeptides: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12.

In other preferred embodiments, the cMet targeting constructs of theinvention include two or more of the following binding polypeptides: SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, and/or SEQ ID NO:29.

In another embodiment, the invention provides a novel method forscreening the KDR targeting constructs for the ability to bind thetarget, and thus, identify multimeric constructs of KDR bindingpolypeptides with improved binding (as determined, for example, bydissociation constants), as compared to binding of the constituentpolypeptides. Additionally, the method of the invention allows for rapiddetermination of whether the multimeric targeting constructs will bestable in the presence of serum in vivo.

Constructs comprising two or more KDR or KDR/VEGF binding polypeptidesshow improved ability to bind the target molecule compared to thecorresponding monomeric binding polypeptides. For instance, as shown inExample 6 below, tetrameric constructs of KDR binding polypeptidesprovided herein showed improved ability to bind KDR-transfected 293Hcells. Combining two or more binding polypeptides in a single molecularconstruct appears to improve the avidity of the construct over themonomeric binding polypeptides a shown by a decrease in K_(D).

In addition, as demonstrated herein, constructs comprising two or morebinding polypeptides specific for different epitopes of KDR and/orKDR/VEGF (e.g., “heteromeric” constructs) were made. Constructscomprising two or more binding polypeptide provided herein are expectedto block multiple sites on KDR or VEGF/KDR. The heteromeric constructsshow superior binding ability over both the corresponding monomers, aswell as tetrameric constructs comprising multiple copies of the samebinding polypeptide. Furthermore, heteromeric constructs comprising twoor more binding peptides specific for different epitopes were also ableto efficiently bind KDR-transfected 293H cells. Thus, inclusion of twoor more binding polypeptides that recognize different epitopes furtherimproves the avidity of the construct for the target molecule, asdemonstrated by a decrease in K_(D).

Heteromeric constructs of the binding polypeptides provided herein showimproved ability to inhibit receptor tyrosine kinase function. Based onexperiments described herein, dimeric and other multimeric constructs ofthe present invention comprising at least two binding polypeptidesspecific for different epitopes of KDR and/or KDR/VEGF are expected toinhibit the function of receptor tyrosine kinases. In particular, suchconstructs are expected to inhibit the function of VEGFR-2/KDR,VEGFR-1/Flt-1 and VEGFR-3/Flt-4. Additionally, heteromultimericconstructs of the invention comprising two or more binding moietiesspecific for different epitopes of cMet and/or cMet/HGF are expected toinhibit the function of receptor tyrosine kinases and, in particular thefunction of cMet.

For the purposes of the present invention, receptor tyrosine kinasefunction can include any one of: oligomerization of the receptor,receptor phosphorylation, kinase activity of the receptor, recruitmentof downstream signaling molecules, induction of genes induction of cellproliferation, induction of cell migration, or combination thereof. Forexample, heteromeric constructs of binding polypeptides provided hereininhibit VEGF-induced KDR receptor inactivation in human endothelialcells, demonstrated by the inhibition of VEGF-induced phosphorylation ofthe KDR receptor. In addition, heteromeric constructs of bindingpeptides provided herein inhibit VEGF-stimulated endothelial cellmigration. As shown herein, targeting two or more distinct epitopes onKDR with a single binding construct greatly improves the ability of theconstruct to inhibit receptor function. Even binding peptides with weakability to block receptor activity can be used to generate heteromericconstructs having improved ability to block VEGF-induced receptorfunction.

Additionally, as further demonstrated herein, constructs comprising twoor more binding polypeptides specific for different epitopes of cMetwere made. Constructs containing two or more cMet binding polypeptideprovided herein are expected to block multiple sites on cMet. Theseheteromeric cMet targeting constructs show superior binding ability overthe corresponding monomers.

Therefore, the present invention is drawn to constructs comprising twoor more binding polypeptides. The multimeric constructs of the presentinvention comprise two or more binding polypeptides, such that at leasttwo of the binding polypeptides in the construct are specific fordifferent epitopes of a target, for example, KDR and/or KDR/VEGF andcMet and/or cMet/HGF. These constructs are also referred to herein as“heteromeric constructs,” “hetermultimers” and/or “heteromultimericconstructs.” The constructs of the present invention can also includeunrelated, or control peptide. The constructs can include two or more,three or more, or four or more binding polypeptides. Based on theteachings provided herein, one of ordinary skill in the art is able toassemble the binding polypeptides provided herein into multimericconstructs and to select multimeric constructs having improvedproperties, such as improved ability to bind the target molecule, orimproved ability to inhibit receptor tyrosine kinase function. Suchmultimeric constructs having improved properties are included in thepresent invention. Furthermore, the methods and teachings providedherein have been shown to allow for the improved binding to a variety ofdifferent targets (e.g., KDR and cMet), thus demonstrating the wideapplicability of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of fluorescent beads to KDR-transfected andmock-transfected cells. Neutravidin-coated beads with the indicatedbiotinylated ligands attached were tested for binding to KDR-expressingand non-expressing 293H cells. Specific binding to KDR was detected forboth P5 (with hydrophilic spacer) and P6. Further details are providedin Example 2.

FIG. 2 shows the percentage inhibition of ¹²⁵I-labeled VEGF binding bypeptides [P6, P4, P5-X-B and P12-X-B) at two different concentrations(30 μM and 0.3 μM) to KDR-transfected 293H cells, as described inExample 3. The results for P6, P4 and P5-X-B are the average of threeexperiments±SD, whereas the result for P12-X-B is based on oneexperiment.

FIG. 3 depicts immunoblots of KDR immunoprecipitates from unstimulated(−V) and VEGF-stimulated (+V) HUVECs which were resolved by SDS-PAGE,blotted, and sequentially probed with anti-phosphotyrosine (“PhosphoKDR”) and anti-KDR (“Total KDR”) antibodies. Activated (phosphorylated)KDR was not detected in unstimulated (−V) HUVECs, but was abundant inimmunoprecipitates from VEGF-stimulated (+V) HUVECs. Reprobing the blotwith anti-KDR demonstrated that comparable amounts of total KDR werepresent in both immunoprecipitates. This figure is representative oftwelve experiments that followed the same protocol.

FIG. 4 depicts immunoblots demonstrating inhibition of KDRphosphorylation (activation) with a neutralizing anti-KDR antibody, asdescribed in Example 4. Immunoprecipitates from unstimulated (−V),VEGF-stimulated (+V), and simultaneously VEGF/anti-KDR (1 μg/mL)(+V+α-KDR)-treated HUVECs were resolved by SDS-PAGE, blotted, andsequentially probed with anti-phosphotyrosine (“Phospho KDR”) andanti-KDR (“Total KDR”) antibodies. As described in Example 4, theneutralizing antibody was able to partially block the VEGF-inducedactivation of KDR.

FIG. 5 depicts immunoblots demonstrating inhibition of KDRphosphorylation (activation) with a KDR-binding peptide (repeatexperiment). Immunoprecipitates from unstimulated (−V), VEGF-stimulated(+V), and a KDR-binding peptide (10 μM) (+V+P10)-treated HUVECs wereresolved by SDS-PAGE, blotted, and sequentially probed withanti-phosphotyrosine (“Phospho KDR”) and anti-KDR (“Total KDR”). Asdescribed in Example 4, the KDR-binding peptide P10 was clearly able topartially block the VEGF-induced activation of KDR at 10 μM.

FIG. 6 depicts binding of Tc-labeled P12-C to mock and KDR transfected293H cells, as described in Example 5.

FIG. 7 depicts specific binding of Tc-labeled P12-C to KDR transfected293H cells, as described in Example 5.

FIG. 8 depicts saturation binding of peptide/Neutravidin HRP complexes,as described in Example 6. FIG. 8A shows the results obtained usingP6-XB and P5-XB. FIG. 8B shows the results obtained using P12-XB andP13-XB. Calculated Kd values were: 10.00 nM (P6-XB), 14.87 nM (P5-XB),4.03 nM (P12-XB) and 1.81 nM (P13-XB).

FIG. 9 depicts binding of peptide/neutravidin HRP complexes (P1-X-B,P5-X-B, P6-XB, P12-XB and P13-XB) to KDR-transfected andmock-transfected 293H cells at a single concentration (5.5 nM), asdescribed in Example 6.

FIG. 10 depicts binding of peptide/neutravidin HRP complexes (P1-XB,P1-B, P5-XB, P5-B, P6-XB and P6-B) to KDR-transfected andmock-transfected 293H cells at a single concentration (2.78 nM), asdescribed in Experiment B of Example 6.

FIG. 11 depicts specific binding (binding to KDR transfected cells minusbinding to mock transfected cells) of peptide/neutravidin HRP complexes(P6-XB, P5-XB, P12-XB and P13-XB) with and without 40% rat serum, asdescribed in Experiment C of Example 6. The concentration ofpeptide/avidin HRP solution was 6.66 nM for P6-XB and P5-XB, 3.33 nM forP12-XB and 2.22 nM for P13-XB.

FIG. 12 shows the binding of peptide/avidin HRP with mock and KDRtransfected cells, plotted as absorbance at 450 nm. The proportions ofcontrol and KDR binding peptides used to form each tetrameric complexare indicated in the legend, for each tested multimer.

FIG. 13 depicts specific binding of a P5-XB/avidin-HRP complex to KDRtransfected cells (background binding to mock-transfected cellssubtracted), plotted as absorbance at 450 nm. Increasing concentrations(as indicated by the X axis) of uncomplexed peptides were added to theassay as indicated in the legend. Only free P5-XB was able to decreasethe binding of the P5-XB/avidin complex to KDR-transfected cells.

FIG. 14 is a graph showing the percentage inhibition of ¹²⁵I-labeledVEGF binding by peptides (P12-XB, D2, D1, D3, and P13-D) at threedifferent concentrations (10 μM, 0.3 μM, and 0.03 μM) to KDR-transfected293H cells. The results are from one experiment carried out intripicate+/−S.D.

FIG. 15 is a photograph showing the ability of D1 to completely blockthe VEGF-induced phosphorylation of KDR in HUVECs at 10 nM and themajority of phosphorylation at 1 nM. Reprobing the blot for total KDR(lower panel) demonstrated that the effects of the tested compounds wasnot due to reduced sample loading. Homodimers composed of the twobinding sequences contained in D1 did not interfere with thephosphorylation at up to 100 nM.

FIG. 16 shows that D1 potently blocks the migration/invasion ofendothelial cells induced by VEGF. Migrating cells were quantitated byfluorescence measurement after staining the migrated cells with afluorescent dye.

FIG. 17 is a graph showing the binding of ¹²⁵I-labeled D5 to mock andKDR transfected 293H cells in the absence and presence of 40% mouseserum.

FIG. 18 is a graph showing the specific binding (KDR-MOCK) of¹²⁵I-labeled D5 to KDR-transfected 293H cells in the absence andpresence of 40% mouse serum.

FIG. 19 is a graph of plasma clearance as percent injected dose per mLversus time.

FIG. 20 shows SE-HPLC profiles of plasma from the Superdex peptidecolumn. Top panel, sample injected; followed by 0 min, 30 min, and 90min. The insert within each panel shows time point, animal number andvolume injected for HPLC analysis.

FIG. 21 is a graph showing the results of testing of KDR peptides inHUVEC proliferation assay. A, D6; B, P12-G; C, PNC-1 (negative control);F, PNC-1 (negative control).

FIG. 22 shows the kinetic analysis of D1 (heterodimer of a truncatedform of P6-D and P12-G) binding to murine KDR-Fc. All sensograms are fitto the bivalent analyte model.

FIG. 23 shows the kinetic analysis of D7 (heterodimer of P5-D and P6-D)binding to murine KDR-Fc. All sensograms are fit to the bivalent analytemodel.

FIG. 24 shows kinetic analysis of fluorescein labeled P12-G binding tomurine KDR-Fc. All sensograms are fit to the 1:1 Langmuir model.

FIG. 25 is a graph showing the specific binding (binding toKDR-transfected cells minus binding to mock-transfected cells) of^(99m)Tc-labeled P12C in the presence and absence of 40% rat serum, asdescribed in Experiment C of Example 6. Results are plotted as specificCPM bound+/−s.d.

FIG. 26 is a graph depicting % inhibition±s.d. of specific ¹²⁵I-VEGFbinding to KDR-transfected cells by PG-1 (squares) D1 (diamonds).

FIG. 27 is a graph depicting % maximum VEGF-stimulated migration±s.d. ofHUVEC cells in the presence of the indicated concentrations of PG-1(diamonds) D1 (squares).

FIG. 28A is a graph depicting the binding of Tc-labeled D10 toKDR-transfected 293H cells as described in Example 18.

FIG. 28B is a graph depicting the lack of binding of Tc-labeled D18 toKDR-transfected 293H cells as described in example 18.Mock=mock-transfected. Trans=KDR-transfected. MS=mouse serum.

FIG. 29 is a graph depicting the binding of Lu-labeled D13 toKDR-transfected 293H cells as described in Example 19.Mock=mock-transfected. Trans=KDR-transfected. MS=mouse serum.

FIG. 30 is a graph summarizing the results of a radiotherapy study withD13 conducted in nude mice implanted with PC3 tumors. Each plotted linerepresents the growth over time for an individual tumor in a treatedmouse, except for the heavy dashed line, which represents the averagetumor growth in a set of untreated mice, as described in Example 20.

FIG. 31 is a graph showing the total binding of complexes of controlpeptide and the test peptides (P30-XB, P31-XB, P32-XB) with¹²⁵I-streptavidin (in the presence of VEGF) to mock-transfected andKDR-transfected cells. Only the complex containing P30-XB showedspecific binding (KDR-mock).

FIG. 32 is a graph showing that D26 (squares) with its glycosylation andmodified spacer is able to block VEGF-stimulated migration even morepotently than D24 (diamonds), which lacks those chemical modifications.

FIG. 33 is a graph showing that TK-1 enhances the potency of D6 inblocking the biological effects of VEGF in a migration assay withcultured HUVECs. Diamonds: D6 alone at the indicated concentrations.Squares: D6 at the indicated concentrations plus 100 nM TK-1 (constant).

FIG. 34 is a graph showing that homodimeric D8 (squares) is less ablethan heterodimeric D17 (diamonds) to block the effects of VEGF in themigration assay as carried out in Example 25.

FIG. 35 is a graph showing cell proliferation data for D6 as describedin Example 31 below.

FIG. 36 shows examples of (A) a C-terminus to C-terminus linked dimer,(B) an N-terminus to C-terminus linked dimer, and (C) an N-terminus toN-terminus linked dimer.

FIG. 37 is a graph showing uptake and retention of bubble contrast inthe tumor up to 30 minutes post injection for suspensions ofphospholipid stabilized microbubbles conjugated to a heteromultimericconstruct.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that compoundshaving two or more binding moieties, wherein at least two of the bindingmoieties bind to different binding sites on the same target, haveunexpected and significantly improved ability to bind the target.Preferably the target is a receptor or a receptor/ligand complex. Theimproved ability of compounds of the invention (variously referred to as“multivalent targeting constructs,” “heterodimers,” “heterotetramers,”“heteromultimers” and/or “heteromultimeric constructs” herein) to bind atarget may be demonstrated by comparison to the ability of anindividual, constituent, binding moiety to bind the target. For example,the binding strength of a heteromultimer of the invention may becompared to the binding strength of one of its monomers. Preferably, aheteromultimer of the invention exhibits an increase in affinity (asdetermined, for example, by dissociation constants), compared to anindividual, constituent monomer.

DEFINITIONS

As used herein, the term “recombinant” is used to describe non-naturallyaltered or manipulated nucleic acids, host cells transfected withexogenous nucleic acids, or polypeptides expressed non-naturally,through manipulation of isolated DNA and transformation of host cells.Recombinant is a term that specifically encompasses DNA molecules whichhave been constructed in vitro using genetic engineering techniques, anduse of the term “recombinant” as an adjective to describe a molecule,construct, vector, transfected cell, polypeptide or polynucleotidespecifically excludes naturally occurring such molecules, constructs,vectors, cells, polypeptides or polynucleotides.

The term “bacteriophage” is defined as a bacterial virus containing aDNA core and a protective shell built up by the aggregation of a numberof different protein molecules. The terms “bacteriophage” and “phage”are used herein interchangeably.

The term “polypeptide” is used to refer to a compound of two or moreamino acids joined through the main chain (as opposed to side chain) bya peptide amide bond (—C(:O)NH—). The term “peptide” is usedinterchangeably herein with “polypeptide” but is generally used to referto polypeptides having fewer than 40, and preferably fewer than 25 aminoacids.

The term “binding” refers to the determination by standard assays,including those described herein, that a binding polypeptide recognizesand binds reversibly to a given target. Such standard assays include,but are not limited to, equilibrium dialysis, gel filtration, and themonitoring of spectroscopic changes that result from binding.

The term “binding polypeptide” as used herein refers to any polypeptidecapable of forming a binding complex with another molecule. Alsoincluded within the definition of “binding polypeptides” arepolypeptides that are modified or optimized as disclosed herein.Specific examples of such modifications are discussed in detail infra,but include substitution of amino acids for those in the parentpolypeptide sequence to optimize properties, obliterate an enzymecleavage site, etc.; C- or N-terminal amino acid substitutions orelongations, e.g., for the purpose of linking the binding polypeptide toa detectable imaging label or other substrate, examples of whichinclude, e.g., addition of a polyhistidine “tail” to assist inpurification; truncations; amide bond changes; translocations;retroinverso peptides; peptoids; retroinversopeptoids; the use ofN-terminal or C-terminal modifications or linkers, such as polyglycineor polylysine segments; alterations to include functional groups,notably hydrazide (—NH—NH₂) functionalities or the C-terminal linker-Gly-Gly-Gly-Lys, to assist in immobilization of binding peptidesaccording to this invention on solid supports or for attachment offluorescent dyes; modifications which effect pharmacokinetics;structural modifications to retain structural features; formation ofsalts to increase water solubility or ease of formulation, and the like.In addition to the detectable labels described further herein, thebinding polypeptides may be linked or conjugated to a radiotherapeuticagent, a cytotoxic agent, a tumorcidal agent or enzyme, a liposome(e.g., loaded with a therapeutic agent, an ultrasound appropriate gas,or both). In addition, binding polypeptides of the invention may bebound or linked to a solid support, such as a well, plate, bead, tube,slide, filter, or dish. Moreover, dimers or multimers of one or morebinding polypeptides may be formed. Such constructs may, for example,exhibit increased ability to bind to the target. All such modifiedpolypeptides are also considered “binding polypeptides” so long as theyretain the ability to bind the targets.

“Homologues” of the binding polypeptides described herein may beproduced using any of the modification or optimization techniquesdescribed herein or known to those skilled in the art. Such homologouspolypeptides will be understood to fall within the scope of the presentinvention and the definition of “binding polypeptides” so long as thesubstitution, addition, or deletion of amino acids or other suchmodification does not eliminate its ability to bind to the target. Theterm “homologous,” as used herein, refers to the degree of sequencesimilarity between two polymers (i.e., polypeptide molecules or nucleicacid molecules). When the same nucleotide or amino acid residue or onewith substantially similar properties (i.e. a conservative substitution)occupies a sequence position in the two polymers under comparison, thenthe polymers are homologous at that position. For example, if the aminoacid residues at 60 of 100 amino acid positions in two polypeptidesequences match or are homologous then the two sequences are 60%homologous. The homology percentage figures referred to herein reflectthe maximal homology possible between the two polymers, i.e., thepercent homology when the two polymers are so aligned as to have thegreatest number of matched (homologous) positions. Polypeptidehomologues within the scope of the present invention will be at least70% and preferably greater than 80% homologous to at least one of thebinding sequences disclosed herein.

“KDR binding polypeptide” is a binding polypeptide that forms a complexin vitro or in vivo with vascular endothelial growth factor receptor-2(or KDR, Flk-1);

“VEGF/KDR complex binding polypeptide” is a binding polypeptide thatforms a complex in vitro or in vivo with a binding complex formedbetween vascular endothelial growth factor (VEGF) and KDR, in particularthe complex of homodimeric VEGF and one or two KDR molecules that isbelieved to form at the surface of endothelial cells duringangiogenesis. Specific examples of KDR and VEGF/KDR binding polypeptidesinclude but are not limited to the peptides presented discussed herein,and in U.S. Ser. No. 60/360,851 and U.S. Ser. No. 60/440,441, both ofwhich are incorporated by reference herein in their entirety, and incopending application U.S. Ser. No. ______, entitled “KDR and VEGF/KDRbinding peptides and their use in diagnosis and therapy,” and includehybrid and chimeric polypeptides incorporating such peptides as well ashomologues.

“cMet binding polypeptide” is a binding polypeptide that forms a complexin vitro or in vivo with the HGF receptor, cMet;

“cMet/HGF complex binding polypeptide” is a binding polypeptide thatforms a complex in vitro or in vivo with a binding complex formedbetween hepatocyte growth factor (HGF) and cMet. Specific examples ofcMet and cMet/HGF binding polypeptides include but are not limited tothe peptides presented discussed herein, and in U.S.S.N. copendingprovisional application U.S. Ser. No. ______, entitled “Peptides thatSpecifically Bind HGF Receptor (cMet) and Uses Thereof,” and includehybrid and chimeric polypeptides incorporating such peptides as well ashomologues.

A “labelling group” or “detectable label,” as used herein, is a group ormoiety capable of generating a signal for diagnostic imaging, such asmagnetic resonance imaging, radioimaging, ultrasound imaging, x-rayimaging, light imaging, or carrying a moiety such as a radioactive metalor other entity that may be used in radiotherapy or other forms oftherapy.

The term “specificity” refers to a binding polypeptide having a higherbinding affinity for one target over another. Binding specificity may becharacterized by a dissociation equilibrium constant (K_(D)) or anassociation equilibrium constant (K_(a)) for the two tested targetmaterials. In a preferred embodiment, binding polypeptides of theinvention have a dissociation constant for a desired target that islower than about 10 μM, more preferably lower than about 1 μM, and mostpreferably less than about 0.5 μM or even lower. The term “KDRspecificity” refers to a KDR binding moiety having a higher affinity forKDR than an irrelevant target. The term “VEGF/KDR specificity” refers toa VEGF/KDR complex binding moiety having a higher affinity for aVEGF/KDR complex than an irrelevant target. In a preferred embodiment,heteromultimers according to the present invention are specific for KDRor the VEGF/KDR complex, and preferably have a dissociation constantthat is lower than about 10 μM, more preferably less than about 1 μM,most preferably less than about 0.5 μM or even lower. The term “cMetspecificity” refers to a cMet binding moiety having a higher affinityfor cMet than an irrelevant target. The term “cMet/HGF specificity”refers to a cMet/HGF complex binding moiety having a higher affinity fora cMet/HGF complex than an irrelevant target. In a preferred embodiment,binding heteromultimers according to the present invention are specificfor cMet or the cMet/HGF complex, and preferably have a dissociationconstant that is lower than about 10 μM, more preferably less than about1 μM, most preferably less than about 0.5 μM or even lower.

The term “patient” as used herein refers to any mammal, especiallyhumans.

The term “pharmaceutically acceptable” carrier or excipient refers to anon-toxic carrier or excipient that may be administered to a patient,together with a compound of this invention, and which does not destroythe pharmacological activity thereof.

The term “target” or “target molecule” refers to any substance that abinding moiety or binding polypeptide can bind to, such as proteins orpolypeptides, cells, receptors, carbohydrates, lipids, etc. As usedherein, “target” also includes a family of receptors, such as, forexample, protein-tyrosine kinase receptors.

The terms “therapeutic agent” or “therapeutic” refer to a compound or anagent having a beneficial, therapeutic or cytotoxic effect in vivo.Therapeutic agents include those compositions referred to as, forexample, bioactive agents, cytotoxic agents, drugs, chemotherapy agents,radiotherapeutic agents, genetic material, etc.

The following common abbreviations are used throughout thisspecification: 9-fluorenylmethyloxycarbonyl (fmoc or Fmoc),1-hydroxybenozotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC),acetic anhydride (Ac₂O),(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),trifluoroacetic acid (TFA), Reagent B(TFA:H₂O:phenol:triisopropylsilane, 88:5:5:2), N,N-diisopropylethylamine(DIEA), O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate(HATU), N-hydroxysuccinimide (NHS), solid phase peptide synthesis(SPPS), dimethyl sulfoxide (DMSO), dichloromethane (DCM),dimethylformamide (DMF), and N-methylpyrrolidinone (NMP).

Dimeric and Multimeric Targeting Constructs of the Invention

The targeting constructs of the invention include two or more bindingmoieties which bind to different binding sites of a single target. Thebinding moieties are specific for different sites on the same target.They may be peptidic, peptidomimetic, etc and include bindingpolypeptides as defined herein. Additionally, binding moieties includesmall binding molecules. In a preferred embodiment the binding moietiescomprise binding polypeptides. These targeting constructs are bydefinition dimeric or multimeric and may be referred to as “multivalenttargeting constructs,” “heterodimers,” “heteromultimers,” or“heteromers.” These dimeric or multimeric constructs exhibit improvedbinding, as compared to a monomeric construct. Where the constructscomprise binding polypeptides, the polypeptide sequences may be attachedat their N- or C-terminus or the N-epsilon nitrogen of a suitably placedlysine moiety (or another function bearing a selectively derivatizablegroup such as a pendant oxyamino or other nucleophilic group), or may bejoined together via one or more linkers employing the appropriateattachment chemistry. This coupling chemistry may include amide, urea,thiourea, oxime, or aminoacetylamide (from chloro or bromo acetamidederivatives), but is not so limited.

Preferred dimers according to the invention can be constructed byconnecting a first binding peptide to a branching group to a firstspacer to a linker to second spacer and finally to the second bindingpeptide. This linking scheme for the dimers can be represented by thefollowing general structure:

A-B-C-D-E-F

where A and F are two different binding peptides which bind to differentsites on the same target, B is a branch group, C and E are spacers, andD is a linker. Suitable spacers and linkers are known in the art and arealso provided in the Examples below. In various embodiments, C, D and/orE may optionally be absent. A reporter moiety or similar group mayoptionally be attached to the dimer via the branch group. The exactarrangement of these components can vary depending, for example, onwhether the peptides are linked from C-terminus to C-terminus, fromN-terminus to C-terminus, or from N-terminus to N-terminus. Examples ofthese different attachment schemes are shown in FIG. 36.

The preparation of dimeric constructs bearing two different bindingpeptides (or two molecules of a particular peptide) and a labellinggroup may be accomplished as described herein, as well as by othermethods known in the art. For example, fully protected binding peptidescan be built up on Ellman-type safety catch resin using automated ormanual Fmoc peptide synthesis protocols. See Backes, B. J., et al., J.Am. Chem. Soc. (1996), 118(12), 3055-6, which is hereby incorporated byreference in its entirety. Separately, using standard methods known inthe art of peptide synthesis (see, e.g., Fields, G. B. et al.,“Principles and Practice of Solid Phase Synthesis” in SyntheticPeptides, A Users Guide, Grant, G. A. ed., W.H. Freeman Co. NY. 1992,Chap. 3 pp 77-183, which is hereby incorporated by reference in itsentirety), a di-lysine derivative can be constructed on 2-chlorotritylresin. See Barlos, K. and Gatos, D. “Convergent Peptide Synthesis” inFmoc Solid Phase Peptide Synthesis, Chan, W. C. and White, P. D. eds,Oxford University Press, New York, 2000, Chap 9: pp 215-228, which ishereby incorporated by reference in its entirety. Liberation of thisderivative from the 2-chlorotrityl resin without removal of theside-chain protecting groups, activation of the carboxyl group, andcoupling to any amine-functionalized labelling group provides adi-lysine derivative whose protected pendant nitrogen atoms may beunmasked to give two free amino groups. The aforementioned safety-catchresin is activated and the desired N-deprotected labellinggroup-functionalized di-lysine derivative is added to the activatedsafety-catch resin. The pendant amino groups are acylated by thecarboxy-terminus of the safety-catch resin-bound peptide which is nowdetached from the resin and an integral part of the di-lysine structure.An excess of the safety-catch resin-bound peptide can be employed toinsure complete reaction of the amino groups of the di-lysine construct.Optimization of the ratio of the reacting partners in this schemeoptimizes the yield. The protecting groups on the binding peptides areremoved employing trifluoroacetic acid based cleavage protocols.

For example, the synthesis of dimeric and multimeric constructs whereintwo or more binding peptides are present in one construct is easilyaccomplished. Orthogonal protection schemes (such as an allyloxycarbonylgroup on one nitrogen and an Fmoc group on the other, or employing theFmoc group in conjunction with the iV-Dde protecting group on the other,for example) can be employed to distinguish the pendant nitrogen atomsof the di-lysine derivatives described above. Unmasking of one of theamino groups, followed by reaction of the resulting product with anactivated safety-catch resin-bound binding peptide as described above,provides a di-lysine construct having a single binding peptide attached.Removal of the second protecting group unmasks the remaining nitrogen.See, e.g., Mellor, S. L. et al. “Synthesis of Modified Peptides” in FmocSolid Phase Peptide Synthesis, Chan, W. C. and White, P. D. eds, OxfordUniversity Press, New York, 2000, Chap 6: pp 169-176, which is herebyincorporated by reference in its entirety. The resulting product may bereacted with a second safety-catch resin bearing a different bindingpeptide to provide a fully-protected heterodimeric construct, whichafter removal of protecting groups with trifluoroacetic acid, providesthe desired material.

Alternatively, a binding peptide is first assembled on a Rink-amideresin by automated or manual peptide coupling methods, usually employingFmoc peptide synthesis protocols. The peptide may possess a C-terminusor N-terminus functionalized with a linker or a linker-labelling groupconstruct that may possess an additional nucleophilic group such as theN^(ε)-amino group of a lysine moiety, for example. Cleavage of theprotecting groups is accomplished by employing trifluoroacetic acid withappropriate modifiers, depending on the nature of the peptide. The fullydeprotected peptide is then reacted with a large excess of abifunctional electrophile such as glutaric acid bis-N-hydroxysuccinimideester (commercially available from Tyger Scientific Inc., 324 StokesAvenue, Ewing, N.J., 08638). The resulting monoamidated,mono-N-hydroxysuccinimidyl ester of glutaric acid is then treated withan additional equivalent of the same peptide, or an equivalent of adifferent binding peptide. Purification of the resulting material byHPLC affords the desired homo- or hetero-dimeric construct bearing asuitable labelling group.

In yet another approach, a modular scheme can be employed to preparedimeric or higher multimeric constructs bearing suitable labellinggroups as defined above. In a simple illustration, fmoc-lysine(iV-Dde)Rink amide resin is treated with piperidine to remove the fmoc moiety.Then a labelling function, such as biotin, 5-carboxyfluorescein orN,N-Dimethyl-Gly-Ser(O-t-Bu)-Cys(Acm)-Gly-OH is coupled to the nitrogenatom. The resin is next treated with hydrazine to remove the iV-Ddegroup. After thorough washing, the resin is treated with cyanuricchloride and a hindered base such as diisopropylethylamine in a suitablesolvent such as DMF, NMP or dichloromethane to provide amonofunctionalized dichlorotriazine bound to the resin. Subsequentsuccessive displacement of the remaining chlorine atoms either by twoequivalents of a binding peptide or one equivalent of a binding peptide,followed by a second binding peptide provides a resin-bound, hetero- orhomo-dimeric, labelling group-functionalized construct. See, e.g.,Falorni, M., et al., Tetrahedron Lett. (1998), 39(41), 7607-7610;Johnson, C. R., et al., Tetrahedron (1998), 54(16), 4097-4106; Stankova,M. and Lebl, M., Mol. Diversity (1996), 2(1/2), 75-80.

As appropriate, the incoming peptides may be protected or unprotected asthe situation warrants. Cleavage of protecting groups is accomplishedemploying trifluoroacetic acid-based deprotection reagents as describedabove and the desired materials are purified by high performance liquidchromatography.

It is understood that in each of these methods, lysine derivatives,ornithine, or 2,3-diamino propionic acid may be serially employed toincrease the multiplicity of the multimers. The use of related, morerigid molecules bearing the requisite number of masked, or orthogonallyprotected nitrogen atoms to act as scaffolds, to vary the distancebetween the binding peptides, and to increase the rigidity of theconstruct (by constraining the motion and relative positions of thebinding peptides relative to each other and the reporter) is entirelywithin the scope of the synthetic methods described herein.

Direct synthesis of the binding polypeptides may be accomplished usingconventional techniques, including solid-phase peptide synthesis,solution-phase synthesis, etc. Solid-phase synthesis is preferred. SeeStewart et al., Solid-Phase Peptide Synthesis (1989), W. H. Freeman Co.,San Francisco; Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963);Bodanszky and Bodanszky, The Practice of Peptide Synthesis(Springer-Verlag, New York 1984), incorporated herein by reference.Polypeptides of the invention may also be prepared commercially bycompanies providing peptide synthesis as a service (e.g., BACHEMBioscience, Inc., King of Prussia, Pa.; Quality Controlled Biochemicals,Inc., Hopkinton, Mass.). Automated peptide synthesis machines, such asmanufactured by Perkin-Elmer Applied Biosystems, also are available.

The polypeptide compound is preferably purified once it has beenisolated or synthesized by either chemical or recombinant techniques.For purification purposes, there are many standard methods that may beemployed, including reverse-phase high-pressure liquid chromatography(RP-HPLC) using an alkylated silica column such as C₄-, C₈- orC₁₈-silica. A gradient mobile phase of increasing organic content isgenerally used to achieve purification, for example, acetonitrile in anaqueous buffer, usually containing a small amount of trifluoroaceticacid. Ion-exchange chromatography can also be used to separate peptidesbased on their charge. The degree of purity of the polypeptide may bedetermined by various methods, including identification of a major largepeak on HPLC. A polypeptide that produces a single peak that is at least95% of the input material on an HPLC column is preferred. Even morepreferable is a polypeptide that produces a single peak that is at least97%, at least 98%, at least 99% or even 99.5% or more of the inputmaterial on an HPLC column.

To ensure that the peptide obtained using any of the techniquesdescribed above is the desired peptide for use in compositions of thepresent invention, analysis of the peptide composition may be carriedout. Such composition analysis may be conducted using high resolutionmass spectrometry to determine the molecular weight of the peptide.Alternatively, the amino acid content of the peptide can be confirmed byhydrolyzing the peptide in aqueous acid, and separating, identifying andquantifying the components of the mixture using HPLC, or an amino acidanalyzer. Protein sequenators, which sequentially degrade the peptideand identify the amino acids in order, may also be used to determine thesequence of the peptide.

For example, binding polypeptides also may be produced using recombinantDNA techniques, utilizing nucleic acids (polynucleotides) encoding thepolypeptides of the invention, and then expressing them recombinantly,i.e., by manipulating host cells by introduction of exogenous nucleicacid molecules in known ways to cause such host cells to produce thedesired binding polypeptides. Such procedures are within the capabilityof those skilled in the art (see Davis et al., Basic Methods inMolecular Biology, (1986)), which is hereby incorporated by reference inits entirety. Recombinant production of short peptides such as thosedescribed herein may not be practical in comparison to direct synthesis,however recombinant means of production may be very advantageous where abinding moiety of this invention is incorporated in a hybrid polypeptideor fusion protein.

In the practice of one embodiment of the present invention, adetermination of the affinity of the heteromultimer or a constituentbinding moiety for the target relative to another protein or target is auseful measure, and is referred to as affinity for the target. Standardassays for quantitating binding and determining affinity includeequilibrium dialysis, equilibrium binding, gel filtration, or themonitoring of numerous spectroscopic changes (such as a change influorescence polarization) that may result from the interaction of thebinding moiety and its target. These techniques or modifications thereofmeasure the concentration of bound and free ligand as a function ofligand (or protein) concentration. The concentration of boundheteromultimer or polypeptide ([Bound]) is related to the concentrationof free heteromultimer or polypeptide ([Free]) and the concentration ofbinding sites for the polypeptide, i.e., on KDR, VEGF/KDR complex, cMet,or the cMet/HGF complex (N), as described in the following equation:

[Bound]=N×[Free]/((1/K _(a))+[Free]).

A solution of the data to this equation yields the association constant,K_(a), a quantitative measure of the binding affinity. The associationconstant, K_(a) is the reciprocal of the dissociation constant, K_(D).The K_(D) is more frequently reported in measurements of affinity. In apreferred embodiment heteromultimers of the invention and constituentbinding polypeptides bind to the target, e.g. KDR, VEGF/KDR complex,cMet or cMet/HGF and have a K_(D) for the target in the range of 1nanomolar (nM) to 100 micromolar (μM) and preferably have K_(D) valuesless than 50 μM, preferably less than 1 μM, more preferably less than 50nM, and most preferably less than 10 nM.

Where heteromultimers are employed as imaging agents, other aspects ofbinding affinity may become more important. For example, such imagingagents operate in a dynamic system in that binding of the imaging agentto the target (such as KDR or VEGF/KDR complex, e.g., on activatedendothelium) is not in a stable equilibrium state throughout the imagingprocedure. For example, when the imaging agent is initially injected,the concentration of imaging agent and of agent-target complex rapidlyincreases. Shortly after injection, however, the circulating (free)imaging agent starts to clear through the kidneys or liver, and theplasma concentration of imaging agent begins to drop. This drop in theconcentration of free imaging agent in the plasma eventually causes theagent-target complex to dissociate. The usefulness of an imaging agentdepends on the difference in rate of agent-target dissociation relativeto the clearing rate of the agent. Ideally, the dissociation rate willbe slow compared to the clearing rate, resulting in a long imaging timeduring which there is a high concentration of agent-target complex and alow concentration of free imaging agent (background signal) in theplasma.

An advantage of heteromultimeric binding compounds, such as those of thepresent invention, is that they generally possess very slow dissociationrates relative to their constituent monomers (see Tissot et al., J.Immunol. Methods 236(1-2):147-165 (2000)). In addition, heteromultimericcompounds capable of binding to two distinct epitopes on a targetmolecule simultaneously can achieve multimeric binding regardless of thedistance between target molecules on the cell surface. Homomultimericbinding compounds, on the other hand, depend on the presence of two ormore target molecules being in close enough proximity such that thehomomultimer can span the distance between them. Thus, theheteromultimeric binding compounds of the present invention areparticularly well suited for binding to receptors and other cell surfacemolecules that are less abundant and therefore more distant from eachother on the cell surface.

Quantitative measurement of dissociation rates may be easily performedusing several methods known in the art, such as fiber optic fluorimetry(see, e.g., Anderson and Miller, Clin. Chem., 34(7):1417-21 (1988)),surface plasmon resonance (see, Malmborg et al., J. Immunol. Methods,198(1):51-7 (1996) and Schuck, Current Opinion in Biotechnology,8:498-502 (1997)), resonant mirror, and grating coupled planarwaveguiding (see, e.g., Hutchinson, Molec. Biotechnology, 3:47-54(1995)). Automated biosensors are commercially available for measuringbinding kinetics: BIAcore surface plasmon resonance sensor (Biacore AB,Uppsala SE), IAsys resonant mirror sensor (Fisons Applied SensorTechnology, Cambridge GB), BIOS-1 grated coupled planar waveguidingsensor (Artificial Sensor Instruments, Zurich CH).

Modification or Optimization of Binding Polypeptides

Modification or optimization of heteromultimers is within the scope ofthe present invention. In particular, modified or optimizedheteromultimers are included within the definition of “heteromultimers”.Similarly, modified or optimized binding polypeptides are includedwithin the definition of “binding polypeptides” and the phrase “KDR andVEGF/KDR complex binding polypeptides” includes modified or optimizedKDR and VEGF/KDR binding polypeptides, and the phrase “cMet and cMet/HGFcomplex binding polypeptides” includes modified or optimized cMet andcMet/HGF binding polypeptides. Specifically, a polypeptide sequence foruse in the heteromultimers of the invention can be modified to optimizeits potency, pharmacokinetic behavior, stability and/or otherbiological, physical and chemical properties.

Substitution of Amino Acid Residues

Substitutions of amino acids within the same class (e.g., substitutingone basic amino acid for another) are well known in the art. Forexample, one can make the following isosteric and/or conservative aminoacid changes in the parent polypeptide sequence with the expectationthat the resulting polypeptides would have a similar or improved profileof the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: Includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from 1-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.Substitution of aromatic-substituted hydrophobic amino acids: Includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theprevious listed aromatic amino acids, illustrative examples of whichare: 2-, 3-, or 4-aminophenylalanine, 2-, 3-, or 4-chlorophenylalanine,2-, 3-, or 4-methylphenylalanine, 2-, 3-, or 4-methoxyphenylalanine,5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or4′-amino-, 2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-,3′-, or 4′-methyl-2-, 3- or 4-biphenylalanine, and 2- or3-pyridylalanineSubstitution of amino acids containing basic functions: Includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀ branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma'-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known activated derivatives such as acidchlorides, active esters, active azolides and related derivatives) andlysine, ornithine, or 2,3-diaminopropionic acid.Substitution of acidic amino acids: Including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, aralkyl, and heteroarylsulfonamides of 2,3-diaminopropionic acid, ornithine or lysine andtetrazole-substituted alkyl amino acids.Substitution of side chain amide residues: Including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.Substitution of hydroxyl containing amino acids: Including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

It is also understood that the amino acids within each of the categorieslisted above may be substituted for another of the same group.

Substitution of Amide Bonds

Another type of modification within the scope of the invention is thesubstitution of amide bonds within the backbone of a bindingpolypeptide. For example, to reduce or eliminate undesired proteolysis,or other degradation pathways which diminish serum stability, resultingin reduced or abolished bioactivity, or to restrict or increaseconformational flexibility, it is common to substitute amide bondswithin the backbone of the peptides with functionality that mimics theexisting conformation or alters the conformation in the manner desired.Such modifications may produce increased binding affinity or improvedpharmacokinetic behavior. It is understood that those knowledgeable inthe art of peptide synthesis can make the following amide bond changesfor any amide bond connecting two amino acids with the expectation thatthe resulting peptides could have the same or improved activity:insertion of alpha-N-methylamides or peptide amide backbone thioamides,removal of the carbonyl to produce the cognate secondary amines,replacement of one amino acid with an aza-aminoacid to producesemicarbazone derivatives, and use of E-olefins and substitutedE-olefins as amide bond surrogates.

Introduction of D-Amino Acids

Another approach within the scope of the invention is the introductionof D-alanine, or another D-amino acid, distal or proximal to a labilepeptide bond. In this case it is also understood to those skilled in theart that such D-amino acid substitutions can, and at times, must bemade, with D-amino acids whose side chains are not conservativereplacements for those of the L-amino acid being replaced. This isbecause of the difference in chirality and hence side-chain orientation,which may result in the accessing of a previously unexplored region ofthe binding site of the target which has moieties of different charge,hydrophobicity, steric requirements, etc., than that serviced by theside chain of the replaced L-amino acid.

Modifications to Improve Pharmacokinetic or Pharmacodynamic Properties

It is also understood that use of the heteromultimeric constructs of theinvention in a particular application may necessitate modifications ofthe peptide or formulations of the peptide to improve pharmacokineticand pharmacodynamic behavior. It is expected that the properties of thepeptide may be changed by attachment of moieties anticipated to bringabout the desired physical or chemical properties. Where theheteromultimer includes binding polypeptides, such moieties affectingthe pharmacokinetic and pharmacodynamic behavior may be appended to thepeptide using acids or amines, via amide bonds or urea bonds,respectively, to the N- or C-terminus of the peptide, or to the pendantamino group of a suitably located lysine or lysine derivative,diaminopropionic acid, ornithine, or other amino acid in the peptidethat possesses a pendant amine group or a pendant alkoxyamino orhydrazine group. The moieties introduced may be groups that arehydrophilic, basic, or nonpolar alkyl or aromatic groups depending onthe peptide of interest and the extant requirements for modification ofits properties.

Glycosylation of Amino Acid Residues

Yet another modification within the scope of the invention is to employglycosylated amino acid residues (e.g. serine, threonine or asparagineresidues), singly or in combination in the either the binding or thelinker moiety or both. Glycosylation, which may be carried out usingstandard conditions, may be used to enhance solubility, alterpharmacokinetics and pharmacodynamics or to enhance binding via aspecific or non-specific interaction involving the glycosidic moiety. Inanother approach glycosylated amino acids such asO-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl) serine orthe analogous threonine derivative (either the D- or L-amino acids) maybe incorporated into the peptide during manual or automated solid phasepeptide synthesis, or in manual or automated solution phase peptidesynthesis. Similarly D- orL-N^(γ)-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-asparaginecan be employed. The use of amino acids glycosylated on a pendantoxygen, nitrogen or sulfur function by the agency of suitablyfunctionalized and activated carbohydrate moieties that can be employedin glycosylation is anticipated. Such carbohydrate functions could bemonosaccharides, disaccharides or even larger assemblies ofoligosaccharides (Kihlberg, Jan. (2000) Glycopeptide synthesis. In: FmocSolid Phase Peptide Synthesis—A Practical Approach (Chan, W. C. andWhite, P. D. Eds) Oxford University Press, New York, N.Y. Chap. 8, pp195-213).

Also anticipated is the appendage of carbohydrate functions to aminoacids by means other than glycosylation via activation of a leavinggroup at the anomeric carbon. Linkage of the amino acid to the glycosideis not limited to the formation of a bond to the anomeric carbon of thecarbohydrate function. Instead, linkage of the carbohydrate moiety tothe amino acid could be through any suitable, sufficiently reactiveoxygen atom, nitrogen atom, carbon atom or other pendant atom of thecarbohydrate function via methods employed for formation ofC-heteroatom, C—C or heteroatom-heteroatom (examples are S—S, O—N, N—N,P—O, P—N) bonds known in the art.

Formation of Salts

It is also within the scope of the invention to form different saltsthat may increase the water solubility or the ease of formulation ofthese peptides. These may include, but are not restricted to,N-methylglucamine (meglumine), acetate, oxalates, ascorbates etc.

Structural Modifications which Retain Structural Features

Yet another modification within the scope of the invention is truncationof cyclic polypeptides. The cyclic nature of many polypeptides of theinvention limits the conformational space available to the peptidesequence, particularly within the cycle. Therefore truncation of thepeptide by one or more residues distal or even proximal to the cycle, ateither the N-terminal or C-terminal region may provide truncatedpeptides with similar or improved biological activity. A unique sequenceof amino acids, even as small as three amino acids, which is responsiblefor the binding activity, may be identified, as noted for RGD peptides.See e.g., E. F. Plow et al., Blood (1987), 70(1), 110-5; A. Oldberg etal., Journal of Biological Chemistry (1988), 263(36), 19433-19436; R.Taub et al., Journal of Biological Chemistry (1989 Jan. 5), 264(1),259-65; A. Andrieux et al., Journal of Biological Chemistry (1989 Jun.5), 264(16), 9258-65; and U.S. Pat. Nos. 5,773,412 and 5,759,996, eachof which is incorporated herein by reference in its entirety.

It has also been shown in the literature that large peptide cycles canbe substantially shortened, eliminating extraneous amino acids, butsubstantially including the critical binding residues. See U.S. Pat. No.5,556,939, which is incorporated herein by reference in its entirety.Shortened cyclic peptides can be formed using disulfide bonds or amidebonds of suitably located carboxylic acid groups and amino groups.

Furthermore, D-amino acids can be added to the peptide sequence tostabilize turn features (especially in the case of glycine). In anotherapproach alpha, beta, gamma or delta dipeptide or turn mimics (such asα, β, γ, or δ turn mimics) some of which are shown in structures 1, 2and 3, below, can be employed to mimic structural motifs and turnfeatures in a peptide and simultaneously provide stability fromproteolysis and enhance other properties such as, for example,conformational stability and solubility (structure 1: Hart et al., J.Org. Chem., 64, 2998-2999 (1999); structure 2: Hanessian et al.,“Synthesis of a Versatile Peptidomimetic Scaffold” in Methods inMolecular Medicine, Vol. 23: Peptidomimetics Protocols, W. M. KazmierskiEd. (Humana Press Inc. Totowa N.J. 1999), Chapter 10, pp. 161-174;structure 3: WO 01/16135.

Substitution of Disulfide Mimetics

Also included within the scope of the invention is the substitution ofdisulfide mimetics for disulfide bonds within the binding polypeptidesof the invention. When disulfide-containing peptides are employed ingenerating heteromultimeric constructs, the disulfide bonds might needto be replaced to avoid certain difficulties that are sometimes posed bythe presence of a disulfide bond. For example, when generatingheteromultimeric ^(99m)Tc (or other radionuclide)-basedradiopharmaceuticals or certain other hetermultimeric constructs thepresence of the disulfide bond can be a significant problem. Theintegrity of the disulfide bond is difficult to maintain duringprocedures designed to incorporate ^(99m)Tc via routes that are reliantupon the reduction of pertechnetate ion and subsequent incorporation ofthe reduced Tc species into substances bearing Tc-compatible chelatinggroups. This is because the disulfide bond is rather easily reduced bythe reducing agents commonly used in kits devised for one-steppreparation of radiopharmaceuticals. Therefore, the ease with which thedisulfide bond can be reduced during Tc chelation may requiresubstitution with mimetics of the disulfide bonds. Accordingly, anothermodification within the scope of the invention is to substitute thedisulfide moiety with mimetics, utilizing the methods disclosed hereinor known to those skilled in the art, while retaining the activity andother desired properties of the binding polypeptides used in theinvention:

1.) Oxime Linker

The oxime moiety has been employed as a linker by investigators in anumber of contexts. Of the most interest is the work by Wahl, F andMutter, M, Tetrahedron Lett. (1996) 37, 6861-6864). The amino acidscontaining an aminoalcohol function (4), and containing an alkoxyaminofunction (5), are incorporated into the peptide chain, not necessarilyat the end of the peptide chain. After formation of the peptide, thesidechain protecting groups are removed. The aldehyde group is unmaskedand an oxime linkage is formed.

2.) Lanthionine Linker

Lanthionines are cyclic sulfides, wherein the disulfide linkage (S—S) isreplaced by a (C—S) linkage. Thus the lability to reduction is far lowerand this linkage should be stable to stannous chloride. Lanthionines maybe prepared by a number of methods.

Preparation of Lanthionines Using Bromoacetylated Peptides

Lanthionines are readily prepared using known methods. See, for example,Robey et al. (Robey, F. A. and Fields, R. L. Anal. Biochem. (1989) 177,373-377) and Inman, et al. (Inman, J. K.; Highet, P. F.; Kolodny, N.;and Robey, F. A. Bioconjugate Chem. (1991) 2, 458-463; Ploinsky, A.Cooney, M. C. Toy-Palmer, A. Osapay, G. and Goodman, M. J. Med. Chem.(1992) 35, 4185-4194; Mayer, J. P.; Zhang, J.; and Liu, C. F. in: Tam,J. P. and Kaumaya, P. T. P. (eds), “Peptides, Frontiers of PeptideScience,” Proceedings of the 15^(th) American Peptide Symposium, June14-19 Nashville, Tenn. Klumer Academic Pub. Boston. pp 291-292;. Wakao,Norihiro; Hino, Yoichi; Ishikawa, Ryuichi. Jpn. Kokai Tokkyo Koho(1995), 7 pp. JP 07300452 A2 19951114 Heisei; JP 95-49692 19950309; JP94-41458 19940311 have published in this area. Preparation of peptidesusing Boc automated peptide synthesis followed by coupling the peptideterminus with bromoacetic acid gives bromoacetylated peptides in goodyield. Cleavage and deprotection of the peptides is accomplished usingHF/anisole. If the peptide contains a cysteine group its reactivity canbe controlled with low pH. If the pH of the medium is raised to 6-7,then either polymerization or cyclization of the peptide takes place.Polymerization is favored at high (100 mg/mL) concentration, whereascyclization is favored at lower concentrations (1 mg/mL), e.g., inScheme 1 below, 6 cyclizes to 7.

Inman et al. demonstrated the use ofN^(α)-(Boc)-N^(ε)-[N-(bromoacetyl)-β-alanyl]-L-lysine as a carrier ofthe bromoacetyl group that could be employed in Boc peptide synthesisthus allowing placement of a bromoacetyl bearing moiety anywhere in asequence. In preliminary experiments they found that peptides with 4-6amino acids separating the bromoacetyl-lysine derivative from a cysteinetend to cyclize, indicating the potential utility of this strategy.

Preparation of Lanthionines Via Cysteine Thiol Addition to Acrylamides

Several variants of this strategy may be implemented. Resin-bound serinecan be employed to prepare the lanthionine ring on resin either using abromination-dehydrobromination-thiol addition sequence or by dehydrationwith disuccinimidyl carbonate followed by thiol addition. Ploinsky etal., M. J. Med. Chem., 35:4185-4194 (1992); Mayer et al., “Peptides,Frontiers of Peptide Science”, in Proceedings of the 15^(th) AmericanPeptide Symposium, Tam & Kaumaya (eds), Jun. 14-19, 1995, Nashville,Term. (Klumer Academic Pub. Boston) pp. 291-292. Conjugate addition ofthiols to acrylamides has also been amply demonstrated and a referenceto the addition of 2-mercaptoethanol to acrylamide is provided. Wakao etal., Jpn. Kokai Tokkyo Koho, JP 07300452 A2 (1995).

3.) Diaryl Ether or Diarylamine Linkage

Diaryl Ether Linkage From Intramolecular Cyclization of Aryl BoronicAcids and Tyrosine

The reaction of arylboronic acids with phenols, amines and heterocyclicamines in the presence of cupric acetate, in air, at ambienttemperature, in dichloromethane using either pyridine or triethylamineas a base to provide unsymmetrical diaryl ethers and the related aminesin good yields (as high as 98%) has been reported. See, Evans et al.,Tetrahedron Lett., 39:2937-2940 (1998); Chan et al., Tetrahedron Lett.,39:2933-2936 (1998); Lam et al., Tetrahedron Lett., 39:2941-2944 (1998).In the case of N-protected tyrosine derivatives as the phenol componentthe yields were also as high as 98%. This demonstrates that amino acidamides (peptides) are expected to be stable to the transformation andthat yields are high. Precedent for an intramolecular reaction exists inview of the facile intramolecular cyclizations of peptides to lactams,intramolecular biaryl ether formation based on the S_(N)Ar reaction andthe generality of intramolecular cyclization reactions under highdilution conditions or on resin, wherein the pseudo-dilution effectmimics high dilution conditions.

4.) Formation of Cyclic Peptides with a Lactam Linkage viaIntramolecular Native Chemical Ligation

Another approach that may be employed involves intramolecularcyclization of suitably located vicinal amino mercaptan functions(usually derived from placement of a cysteine at a terminus of thelinear sequence or tethered to the sequence via a side-chain nitrogen ofa lysine, for example) and aldehyde functions to provide thiazolidineswhich result in the formation of a bicyclic peptide, one ring of whichis that formed by the residues in the main chain, and the second ringbeing the thiazolidine ring. Scheme 2, above, provides an example. Therequired aldehyde function can be generated by sodium metaperiodatecleavage of a suitably located vicinal aminoalcohol function, which canbe present as an unprotected serine tethered to the chain by appendageto a side chain amino group of a lysine moiety. In some cases, therequired aldehyde function is generated by unmasking of a protectedaldehyde derivative at the C-terminus or the N-terminus of the chain. Anexample of this strategy is found in: Botti, P.; Pallin, T. D. and Tam,J. P. J. Am. Chem. Soc. 1996, 118, 10018-10034.

5.) Lactams Based on Intramolecular Cyclization of Pendant Amino Groupswith Carboxyl Groups on Resin

Macrocyclic peptides can be prepared by lactam formation by either headto tail or by pendant group cyclization. The basic strategy is toprepare a fully protected peptide wherein it is possible to removeselectively an amine protecting group and a carboxy protecting group.Orthogonal protecting schemes have been developed. Of those that havebeen developed, the allyl, trityl and Dde methods have been employedmost. See, Mellor et al., “Synthesis of Modified Peptides,” in FmocSolid Phase Synthesis: A Practical Approach, White and Chan (eds)([Oxfoerd University Press, New York, 2000]), Chapt. 6, pp. 169-178. TheDde approach is of interest because it utilizes similar protectinggroups for both the carboxylic acid function (Dmab ester) and the aminogroup (Dde group). Both are removed with 2-10% hydrazine in DMF atambient temperature. Alternatively, the Dde can be used for the aminogroup and the allyl group can be used for the carboxyl.

A lactam function, available by intramolecular coupling via standardpeptide coupling reagents (such as HATU, PyBOP etc), could act as asurrogate for the disulfide bond. The Dde/Dmab approach is shown inScheme 3a, below.

Thus, a linear sequence containing, for example, the Dde-protectedlysine and Dmab ester may be prepared on a Tentagel-based Rink amideresin at low load (˜0.1-0.2 mmol/g). Deprotection of both functions withhydrazine is then followed by on-resin cyclization to give the desiredproducts.

In the allyl approach, shown in Scheme 3b, the pendant carboxyl which isto undergo cyclization is protected as an allyl ester and the pendantamino group is protected as an alloc group. On resin, both areselectively unmasked by treatment with palladium tris-triphenylphosphinein the presence of N-methylmorpholine and acetic acid in DMF. Residualpalladium salts are removed using sodium diethyldithiocarbamate in thepresence of DIEA in DMF, followed by subsequent washings with DMF. Thelactam ring is then formed employing HATU/HOAt in the presence ofN-methylmorpholine. Other coupling agents can be employed as describedabove. The processing of the peptide is then carried out as describedabove to provide the desired peptide lactam.

Subsequently cleavage from resin and purification may also be carriedout. For functionalization of the N-terminus of the peptide, it isunderstood that amino acids, such astrans-4-(iV-Dde)methylaminocyclohexane carboxylic acid,trans-4-(iV-Dde)methylaminobenzoic acid, or their alloc congeners couldbe employed. Yet another approach is to employ the safety catch methodto intramolecular lactam formation during cleavage from the resin.

6.) Cyclic Peptides Based on Olefin Metathesis

The Grubbs reaction (Scheme 4, below) involves themetathesis/cyclization of olefin bonds and is illustrated as shownbelow. See, Schuster et al., Angewandte. Chem. Int. Edn Engl.,36:2036-2056 (1997); Miller et al., J. Am. Chem. Soc., 118:9606-9614(1996).

It is readily seen that, if the starting material is a diolefin (16),the resulting product will be cyclic compound 17. The reaction has infact been applied to creation of cycles from olefin-functionalizedpeptides. See, e.g., Pernerstorfer et al., Chem. Commun., 20:1949-50(1997); Covalent capture and stabilization of cylindrical β-sheetpeptide assemblies, Clark et al., Chem. Eur. J., 5(2):782-792 (1999);Highly efficient synthesis of covalently cross-linked peptide helices byring-closing metathesis, Blackwell et al., Angew. Chem., Int. Ed.,37(23):3281-3284 (1998); Synthesis of novel cyclic protease inhibitorsusing Grubbs olefin metathesis, Ripka et al., Med. Chem. Lett.,8(4):357-360 (1998); Application of Ring-Closing Metathesis to theSynthesis of Rigidified Amino Acids and Peptides, Miller et al., J. Am.Chem. Soc., 118(40):9606-9614 (1996); Supramolecular Design by CovalentCapture, Design of a Peptide Cylinder via Hydrogen-Bond-PromotedIntermolecular Olefin Metathesis, Clark et al., J. Am. Chem. Soc.,117(49):12364-12365 (1995); Synthesis of Conformationally RestrictedAmino Acids and Peptides Employing Olefin Metathesis, Miller et al., J.Am. Chem. Soc., 117(21):5855-5856 (1995). One can prepare eitherC-allylated amino acids or possibly N-allylated amino acids and employthem in this reaction in order to prepare carba-bridged cyclic peptidesas surrogates for disulfide bond containing peptides.

One may also prepare novel compounds with olefinic groups.Functionalization of the tyrosine hydroxyl with an olefin-containingtether is one option. The lysine ε-amino group may be another optionwith appendage of the olefin-containing unit as part of an acylatingmoiety, for example. If instead the lysine side chain amino group isalkylated with an olefin containing tether, it can still function as apoint of attachment for a reporter as well. The use of 5-pentenoic acidas an acylating agent for the lysine, ornithine, or diaminopropionicside chain amino groups is another possibility. The length of theolefin-containing tether can also be varied in order to explorestructure activity relationships.

Manipulation of Peptide Sequences

Other modifications within the scope of the invention includemanipulations of peptide sequences which can be expected to yieldpeptides with similar or improved biological properties. These includeamino acid translocations (swapping amino acids in the sequence), use ofretroinverso peptides in place of the original sequence or a modifiedoriginal sequence, peptoids, retro-inverso peptoid sequences, andsynthetic peptides. Structures wherein specific residues are peptoidinstead of peptidic, which result in hybrid molecules, neithercompletely peptidic nor completely peptoid, are contemplated as well.

Linkers

Additionally, modifications within the invention include introduction oflinkers or spacers between the targeting sequence of the binding moietyor binding polypeptide and the detectable label or therapeutic agent.For example, use of such linkers/spacers may improve the relevantproperties of the binding peptides (e.g. increase serum stability,etc.). These linkers may include, but are not restricted to, substitutedor unsubstituted alkyl chains, polyethylene glycol derivatives, aminoacid spacers, sugars, or aliphatic or aromatic spacers common in theart.

For example, suitable linkers include homobifunctional andheterobifunctional cross-linking molecules. The homobifunctionalmolecules have at least two reactive functional groups, which are thesame. The reactive functional groups on a homobifunctional moleculeinclude, for example, aldehyde groups and active ester groups.Homobifunctional molecules having aldehyde groups include, for example,glutaraldehyde and subaraldehyde.

Homobifunctional linker molecules having at least two active ester unitsinclude esters of dicarboxylic acids and N-hydroxysuccinimide. Someexamples of such N-succinimidyl esters include disuccinimidyl suberateand dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonicacid and bis-sulfonate salts such as their sodium and potassium salts.

Heterobifunctional linker molecules have at least two different reactivegroups. Some examples of heterobifunctional reagents containing reactivedisulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate(Carlsson et al., 1978, Biochem J. 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidylm-maleimidobenzoate. Other heterobifunctional molecules includesuccinimidyl 3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-5N-hydroxy-succinimide ester.

Furthermore, linkers which are combinations of the molecules and/ormoieties described above, can also be employed to confer specialadvantage to the properties of the peptide. Lipid molecules with linkersmay be attached to allow formulation of ultrasound bubbles, liposomes orother aggregation based constructs. Such constructs could be employed asagents for targeting and delivery of a diagnostic reporter, atherapeutic agent (e.g. a chemical “warhead” for therapy), or acombination of these.

Uses of Heteromultimeric Constructs

Heteromultimeric constructs of the present invention can be used in amultitude of applications, including immunoassays (e.g., ELISA), aspharmaceuticals useful for treatments of various diseases, as well as inin vivo diagnostic and therapeutic uses. For example, theheteromultimeric constructs described herein will be extremely usefulfor detection and/or imaging of target containing tissue in vitro or invivo. For example, KDR or VEGF/KDR complex binding heteromultimericconstructs will be extremely useful for detection and/or imaging of KDRor VEGF/KDR complex containing tissue, and particularly for detectionand/or imaging of sites of angiogenesis, in which VEGF and KDR areintimately involved, as explained above. Any suitable method of assayingor imaging KDR or VEGF/KDR complex may be employed. Similarly, cMet orHGF/cMet complex binding heteromultimeric constructs will be extremelyuseful for detection and/or imaging of cMet or HGF/cMet complexcontaining tissue, and particularly for detection and/or imaging tumorsor other sites of hyperproliferation, in which HGF and cMet areintimately involved, as explained above. Any suitable method of assayingor imaging cMet or HGF/cMet complex may be employed.

The compounds of the invention also have utility in the treatment of avariety of disease states, whether used alone or in combination withanother therapeutic agent. For example, as discussed, a compound of theinvention that inhibits a biological process that contributes to adisease state may itself be used as a therapeutic or pharmaceuticalcomposition. Alternatively (or in combination), a compound of theinvention may include one or more additional therapeutic agents. In oneembodiment, the invention includes heteromultimers including KDR orVEGF/KDR complex binding moieties which may themselves be used astherapeutics or may be used to localize one or more therapeutic agents(e.g. a chemotherapeutic, a radiotherapeutic, genetic material, etc.) toKDR expressing cells, including sites of angiogenesis, or thoseassociated with a number of pathogens. In another embodiment, theinvention includes heteromultimers including cMet or HGF/cMet complexbinding moieties which may themselves be used as therapeutics or may beused to localize one or more therapeutic agents (e.g. achemotherapeutic, a radiotherapeutic, genetic material, etc.) to cMetexpressing cells, including tumors, sites of hyperproliferation or sitesof angiogenesis.

The heteromultimeric constructs of the present invention areparticularly useful as therapeutic agents for treating conditions thatinvolve endothelial cells. Because an important function of endothelialcells is angiogenesis, or the formation of blood vessels, theheteromultimers of the invention are particularly useful for treatingconditions that involve angiogenesis include, for example, solid tumors,tumor metastases and benign tumors. Such tumors and related disordersare well known in the art and include, for example, melanoma, centralnervous system tumors, neuroendocrine tumors, sarcoma, multiple myelomaas well as cancer of the breast, lung, prostate, colon, head & neck, andovaries. Additional tumors and related disorders are listed in Table 1of U.S. Pat. No. 6,025,331, issued Feb. 15, 2000 to Moses, et al., theteachings of which are incorporated herein by reference. Benign tumorsinclude, for example, hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas. Other relevant diseases that involveangiogenesis include for example, rheumatoid arthritis, psoriasis, andocular disease, such as diabetic retinopathy, retinopathy ofprematurity, macular degeneration, corneal graft rejection, neovascularglaucoma, retrolental fibroplasias, rebeosis, Osler-Webber syndrome,myocardial angiogenesis, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma and wound granulation. Other relevantdiseases or conditions that involve blood vessel growth includeintestinal adhesions, atherosclerosis, scleroderma, and hypertropicscars, and ulcers. Furthermore, the heteromultimers of the presentinvention can be used to reduce or prevent uterine neovascularizationrequired for embryo implantation, for example, as a birth control agent.

For detection of the target in solution, a heteromultimer according tothe invention can be detectably labeled, e.g., fluorescently labeled,enzymatically labeled, or labeled with a radionuclide or paramagneticmetal or attached to bubbles, then contacted with the solution, andthereafter formation of a complex between the heteromultimer and thetarget can be detected. As an example, a fluorescently labeled KDR orVEGF/KDR complex binding heteromultimeric construct may be used for invitro KDR or VEGF/KDR complex detection assays, wherein theheteromultimeric construct is added to a solution to be tested for KDRor VEGF/KDR complex under conditions allowing binding to occur. Thecomplex between the fluorescently labeled KDR or VEGF/KDR complexbinding heteromultimer and KDR or VEGF/KDR complex target can bedetected and quantified by measuring the increased fluorescencepolarization arising from the KDR or VEGF/KDR complex-boundheteromultimer relative to that of the free heteromultimer.Heteromultimers comprising cMet binding moieties may be used similarly.

Alternatively, a sandwich-type “ELISA” assay may be used, wherein aheteromultimeric construct is immobilized on a solid support such as aplastic tube or well, then the solution suspected of containing thetarget is contacted with the immobilized heteromultimeric construct,non-binding materials are washed away, and complexed target is detectedusing a suitable detection reagent, such as a monoclonal antibodyrecognizing the target. The monoclonal antibody is detectable byconventional means known in the art, including being detectably labeled,e.g., radiolabeled, conjugated with an enzyme such as horseradishperoxidase and the like, or fluorescently labeled.

For example, for detection or purification of soluble target in or froma solution, heteromultimers of the invention can be immobilized on asolid substrate such as a chromatographic support or other matrixmaterial, then the immobilized heteromultimer can be loaded or contactedwith the solution under conditions suitable for formation of aheteromultimer:target complex. The non-binding portion of the solutioncan be removed and the complex may be detected, e.g., using an antibodyagainst the target, such as an anti-binding polypeptide antibody (e.g.,anti-KDR, anti-VEGF/KDR complex, anti-cMet, or anti-cMet/HGF complexantibody), or the heteromultimer:target complex may be released from thebinding moiety at appropriate elution conditions.

The biology of angiogenesis and the roles of VEGF and KDR in initiatingand maintaining it have been investigated by many researchers andcontinues to be an active field for research and development. Infurtherance of such research and development, a method of purifying bulkamounts of KDR or VEGF/KDR complex in pure form is desirable, and theKDR and VEGF/KDR complex heteromultimers described herein are especiallyuseful for that purpose, using the general purification methodologydescribed above. Similarly, the biology of tumors and otherhyperproliferative tissue and the roles of cMet and HGF in initiatingand maintaining these have been investigated by many researchers andcontinues to be an active field for research and development. Infurtherance of such research and development, a method of purifying bulkamounts of cMet or HGF/cMet complex in pure form is desirable, and thecMet or HGF/cMet complex heteromultimers described herein are especiallyuseful for that purpose, using the general purification methodologydescribed above.

Diagnostic Imaging

Appropriately labeled heteromultimeric constructs of the presentinvention may be used in in vivo diagnostic applications to imagespecific tissues or cellular disorders. A particularly preferred use forthe heteromultimeric constructs according to the present invention isfor creating visually readable images of target expressing or containingtissue. For this embodiment, the heteromultimers of the invention areconjugated with a label appropriate for diagnostic detection, optionallyvia a linker Suitable linkers can be substituted or unsubstituted alkylchains, amino acid chains (e.g., polyglycine), polyethylene glycols,polyamides, and other simple polymeric linkers known in the art.Preferably, a heteromultimer exhibiting much greater specificity for thetarget than for other serum proteins is conjugated or linked to a labelappropriate for the detection methodology to be employed. For example,heteromultimers of the invention may be conjugated with or without alinker to a paramagnetic chelate suitable for magnetic resonance imaging(MRI), with a radiolabel suitable for x-ray, PET or scintigrapic imaging(including if necessary a chelator, such as those described herein, fora radioactive metal) with an ultrasound contrast agent (e.g. astabilized microbubble, a microballoon, a microsphere or what has beenreferred to as a gas filled “liposome”) suitable for ultrasounddetection, or with an optical imaging dye.

For example, KDR or VEGF/KDR complex binding heteromultimeric constructsof the invention or cMet or HGF complex binding heteromultimericconstructs of the invention may be used to image neoplastic tumors,which require angiogenesis for survival and metastasis, or other sitesof angiogenic activity. In this embodiment, heteromultimeric constructsincluding KDR and VEGF/KDR complex binding polypeptides or cMet orHGF/cMet complex binding polypeptides are converted to imaging reagentsby conjugation with a label appropriate for diagnostic detection,optionally via a linker, as described herein.

In general, the technique of using a detectably labeled heteromultimericconstruct is based on the premise that the label generates a signal thatis detectable outside the patient's body. For example, in oneembodiment, when a detectably labeled heteromultimer of the invention isadministered to the patient in which angiogenesis, e.g., due to a tumor,is occurring, the high affinity of the KDR or VEGF/KDR complex bindingmoieties included in the heteromultimeric constructs for KDR or VEGF/KDRcomplex causes the heteromultimeric construct to bind to the site ofangiogenesis and accumulate label at the site of angiogenesis.Sufficient time is allowed for the labeled heteromultimeric construct tolocalize at the site of angiogenesis. The signal generated by thelabeled peptide is detected by a scanning device which will varyaccording to the type of label used, and the signal is then converted toan image of the site of angiogenesis.

In another embodiment, rather than directly labelling a heteromultimerof the invention with a detectable label or radiotherapeutic construct,heteromultimers of the invention can be conjugated with for example,avidin, biotin, or an antibody or antibody fragment that will bind thedetectable label or radiotherapeutic. For example, in one embodiment,heteromultimers can be conjugated to streptavidin or avidin for in vivobinding to target-containing or expressing cells. After the unboundheteromultimer has cleared from the body, a biotinylated detectablelabel or radiotherapeutic construct (e.g. a chelate molecule complexedwith a radioactive metal) can be infused which will rapidly concentrateat the site where the targeting construct is bound. This approach insome situations can reduce the time required after administering thedetectable label until imaging can take place. It can also increasesignal to noise ratio in the target site, and decrease the dose of thedetectable label or radiotherapeutic construct required. This isparticularly useful when a radioactive label or radiotherapeutic is usedas the dose of radiation that is delivered to normal butradiation-sensitive sites in the body, such as bone-marrow, kidneys, andliver is decreased. This approach, sometimes referred to aspre-targeting or two-step, or three-step approaches was reviewed by S.F. Rosebrough (Q. J. Nucl. Med. 40:234-251; 1996, incorporated byreference herein). In a preferred embodiment, heteromultimericconstructs including KDR or VEGF/KDR binding moieties are used. Inanother preferred embodiment, heteromultimeric constructs including cMetor HGF/cMet binding moieties are used.

A. Magnetic Resonance Imaging

The heteromultimers of the present invention may advantageously beconjugated with one or more paramagnetic metal chelates in order to forma contrast agent for use in MRI. Preferred paramagnetic metal ions haveatomic numbers 21-29, 42, 44, or 57-83. This includes ions of thetransition metal or lanthanide series which have one, and morepreferably five or more, unpaired electrons and a magnetic moment of atleast 1.7 Bohr magneton. Preferred paramagnetic metals include, but arenot limited to, chromium (III), manganese (II), manganese (III), iron(II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium(III), neodymium (III), samarium (III), gadolinium (III), terbium (III),dysprosium (III), holmium (III), erbium (III), europium (III) andytterbium (III). Additionally, heteromultimers of the present inventionmay also be conjugated with one or more superparamagnetic particles.

Gd(III) is particularly preferred for MRI due to its high relaxivity andlow toxicity, and the availability of only one biologically accessibleoxidation state. Gd(III) chelates have been used for clinical andradiologic MR applications since 1988, and approximately 30% of MR examscurrently employ a gadolinium-based contrast agent.

One skilled in the art will select a metal according to dose required todetect target containing tissue and considering other factors such astoxicity of the metal to the subject. See, Tweedle et al., MagneticResonance Imaging (2nd ed.), vol. 1, Partain et al., eds. (W.B. SaundersCo. 1988), pp. 796-7. Generally, the desired dose for an individualmetal will be proportional to its relaxivity, modified by thebiodistribution, pharmacokinetics and metabolism of the metal. Thetrivalent cation, Gd³⁺ is particularly preferred for MRI contrastagents, due to its high relaxivity and low toxicity, with the furtheradvantage that it exists in only one biologically accessible oxidationstate, which minimizes undesired metabolization of the metal by apatient. Another useful metal is Cr³⁺, which is relatively inexpensive.

The paramagnetic metal chelator is a molecule having one or more polargroups that act as a ligand for, and complex with, a paramagnetic metal.Suitable chelators are known in the art and include acids with methylenephosphonic acid groups, methylene carbohydroxamine acid groups,carboxyethylidene groups, or carboxymethylene groups. Examples ofchelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10 teraazacyclododecanetriacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-Cl-EHPG, 5Br-EHPG,5-Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) andderivatives thereof; the class of macrocyclic compounds which contain atleast 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (O and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). Apreferred chelator for use in the present invention is DTPA. Examples ofrepresentative chelators and chelating groups contemplated by thepresent invention are described in WO 98/18496, WO 86/06605, WO91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473,PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S. Pat. No. 5,474,756,U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6,143,274, each of which ishereby incorporated by reference in its entirety. Use of the chelateDO3A is particularly preferred.

In one embodiment of the present invention, the chelator(s) of the MRIcontrast agent is coupled to a heteromultimer, such as, for example onecomprised of KDR or VEGF/KDR complex binding polypeptides or cMet orHGF/cMet complex binding polypeptides. The positioning of the chelate(s)should be selected so as not to interfere with the binding affinity orspecificity of the heteromultimeric construct. Preferably, thechelate(s) will be appended either to the N-terminus or the C-terminus,however the chelate(s) may also be attached anywhere within thesequence. In preferred embodiments, a chelator having a free centralcarboxylic acid group (e.g., DTPA-Asp(β-COOH)-OtBu) makes it easy toattach at the N-terminus of a binding peptide by formation of an amidebond. The chelate(s) could also be attached at the C-terminus with theaid of a linker. Alternatively, isothiocyanate conjugation chemistrycould be employed as a way of linking the appropriate isothiocyantegroup bearing DTPA to a free amino group anywhere within the peptidesequence.

For example, the heteromultimer can be bound directly or covalently tothe metal chelator(s) (or other detectable label), or it may be coupledor conjugated to the metal chelator(s) using a linker, which may be,without limitation, amide, urea, acetal, ketal, double ester, carbonyl,carbamate, thiourea, sulfone, thioester, ester, ether, disulfide,lactone, imine, phosphoryl, or phosphodiester linkages; substituted orunsubstituted saturated or unsaturated alkyl chains; linear, branched,or cyclic amino acid chains of a single amino acid or different aminoacids (e.g., extensions of the N- or C-terminus of the bindingmoieties); derivatized or underivatized polyethylene glycol,polyoxyethylene, or polyvinylpyridine chains; substituted orunsubstituted polyamide chains; derivatized or underivatized polyamine,polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol),polyglycerol, or oligosaccharide (e.g., dextran) chains; alternatingblock copolymers; malonic, succinic, glutaric, adipic and pimelic acids;caproic acid; simple diamines and dialcohols; any of the other linkersdisclosed herein; or any other simple polymeric linkers known in the art(see, e.g., WO 98/18497, WO 98/18496). Preferably the molecular weightof the linker can be tightly controlled. The molecular weights can rangein size from less than 100 to greater than 1000. Preferably themolecular weight of the linker is less than 100. In addition, it may bedesirable to utilize a linker that is biodegradable in vivo to provideefficient routes of excretion for the imaging reagents of the presentinvention. Depending on their location within the linker, suchbiodegradable functionalities can include ester, double ester, amide,phosphoester, ether, acetal, and ketal functionalities.

In general, known methods can be used to couple the metal chelate and aheteromultimer of the invention using such linkers. See, e.g., WO95/28967, WO 98/18496, WO 98/18497 and discussion therein. For example,a heteromultimer can be linked through the N- or C-terminus of acomponent binding moiety via an amide bond, for example, to a metalcoordinating backbone nitrogen of a metal chelate or to an acetate armof the metal chelate itself. The present invention contemplates linkingof the chelate(s) on any position, provided the metal chelate retainsthe ability to bind the metal tightly in order to minimize toxicity.Similarly, a component binding moiety of a heteromultimer may bemodified or elongated in order to generate a locus for attachment to ametal chelate, provided such modification or elongation does noteliminate its ability to bind the target.

MRI contrast reagents prepared according to the disclosures herein maybe used in the same manner as conventional MRI contrast reagents. Whenimaging target containing tissue such as, for example, a site ofangiogenesis, certain MR techniques and pulse sequences may be preferredto enhance the contrast of the site to the background blood and tissues.These techniques include (but are not limited to), for example, blackblood angiography sequences that seek to make blood dark, such as fastspin echo sequences (see, e.g., Alexander et al., Magnetic Resonance inMedicine, 40(2): 298-310 (1998)) and flow-spoiled gradient echosequences (see, e.g., Edelman et al., Radiology, 177(1): 45-50 (1990)).These methods also include flow independent techniques that enhance thedifference in contrast, such as inversion-recovery prepared orsaturation-recovery prepared sequences that will increase the contrastbetween target containing tissue, such as an angiogenic tumor, andbackground tissues. Finally, magnetization transfer preparations mayalso improve contrast with these agents (see, e.g., Goodrich et al.,Investigative Radiology, 31(6): 323-32 (1996)).

The labeled reagent is administered to the patient in the form of aninjectable composition. The method of administering the MRI contrastagent is preferably parenterally, meaning intravenously,intraarterially, intrathecally, interstitially, or intracavitarilly. Forimaging active angiogenesis, intravenous or intraarterial administrationis preferred.

For MRI, it is contemplated that the subject will receive a dosage ofcontrast agent sufficient to enhance the MR signal at the target (e.g. asite of angiogenesis) at least 10%. After injection of theheteromultimeric construct including the MRI reagent, the patient isscanned in the MRI machine to determine the location of any sitescontaining the target. In therapeutic settings, upon targetlocalization, a cytotoxic or therapeutic agent can be immediatelyadministered, if necessary, and the patient can be subsequently scannedto visualize the therapeutic effect.

In a preferred embodiment, heteromultimers including KDR or VEGF/KDRcomplex binding moieties are conjugated to one or more paramagneticmetal chelates or one or more superparamagnetic particles, optionallyvia a linker. In another preferred embodiment, heteromultimericconstructs including cMet or HGF/cMet complex binding moieties are used.Such heteromultimeric constructs are complexed with one or moreparamagnetic metal and administered in a dose sufficient to enhance theMR signal at the site of angiogenesis at least 10%. After injection, thepatient is scanned to determine the location of any sites ofangiogenesis (e.g. angiogenic tumors, etc.) or hyperproliferativetissue. If necessary, upon location of an angiogenic orhyperproliferative site, an anti-angiogenic or tumoricidal agent, suchas, for example, an inhibitor of VEGF (or VEGF activation of KDR) may beadministered. If necessary, the patient may be scanned again tovisualize/track the tumor regression, arrest of angiogenesis, etc.

B. Ultrasound Imaging

When ultrasound is transmitted through a substance, the acousticproperties of the substance will depend upon the velocity of thetransmissions and the density of the substance. Changes in the acousticproperties will be most prominent at the interface of differentsubstances (solids, liquids, gases). Ultrasound contrast agents areintense sound wave reflectors because of the acoustic differencesbetween the agent and the surrounding tissue. Gas containing or gasgenerating ultrasound contrast agents are particularly useful because ofthe acoustic difference between liquid (e.g., blood) and thegas-containing or gas generating ultrasound contrast agent. Because oftheir size, ultrasound contrast agents comprising microbubbles,microballoons, and the like may remain for a longer time in the bloodstream after injection than other detectable moieties; thus a targetedultrasound agent may demonstrate superior imaging of tissue expressingor containing the target.

In this aspect of the invention, the heteromultimeric constructs mayinclude a material that is useful for ultrasound imaging. For example,heteromultimers of the invention may be linked to materials employed toform vesicles (e.g., microbubbles, microballoons, microspheres, etc.),or emulsions containing a liquid or gas which functions as thedetectable label (e.g., an echogenic gas or material capable ofgenerating an echogenic gas). Materials for the preparation of suchvesicles include surfactants, lipids, sphingolipids, oligolipids,phospholipids, proteins, polypeptides, carbohydrates, and synthetic ornatural polymeric materials. See e.g. WO 98/53857, WO 98/18498, WO98/18495, WO 98/18497, WO 98/18496, and WO 98/18501 incorporated hereinby reference in their entirety.

For contrast agents comprising suspensions of stabilized microbubbles (apreferred embodiment), phospholipids, and particularly saturatedphospholipids are preferred. The preferred gas-filled microbubbles canbe prepared by means known in the art, such as, for example, by a methoddescribed in any one of the following patents: EP 554213, U.S. Pat. No.5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No.5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833,U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No.5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat.No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S.Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No.5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S.Pat. No. 5,908,610, each of which is incorporated by reference herein inits entirety. In a preferred embodiment, at least one of thephospholipid moieties has the structure of formula 18 or formula 19shown below and described in U.S. Pat. No. 5,686,060, which is hereinincorporated by reference in its entirety.

Examples of suitable phospholipids include esters of glycerol with oneor two molecules of fatty acids (the same or different) and phosphoricacid, wherein the phosphoric acid residue is in turn bonded to ahydrophilic group, such as choline, serine, inositol, glycerol,ethanolamine, and the like groups. Fatty acids present in thephospholipids are in general long chain aliphatic acids, typicallycontaining from 12 to 24 carbon atoms, preferably from 14 to 22, thatmay be saturated or may contain one or more unsaturations. Examples ofsuitable fatty acids are lauric acid, myristic acid, palmitic acid,stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid,and linolenic acid. Mono esters of phospholipid are also known in theart as the “lyso” forms of the phospholipids.

Further examples of phospholipids are phosphatidic acids, i.e. thediesters of glycerol-phosphoric acid with fatty acids, sphingomyelins,i.e. those phosphatidylcholine analogs where the residue of glyceroldiester with fatty acids is replaced by a ceramide chain, cardiolipins,i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid,gangliosides, cerebrosides, etc.

As used herein, the term phospholipids includes either naturallyoccurring, semisynthetic or synthetically prepared products that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins.

Examples of synthetic phospholipids are e.g.,dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”),diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine(“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatid-ylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts,distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts,distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid(“DAPA”) and its alkali metal salts, dimyristoylphosphatidyl-ethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine(“DPPE”), distearoyl phosphatidyl-ethanolamine (“DSPE”), dimyristoylphosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”),dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin(“DPSP”), and distearoyl sphingomyelin (“DSSP”).

Other preferred phospholipids include dipalmitoylphosphatidylcholine,dipalmitoylphosphatidic acid and dipalmitoylphosphatidylserine. Thecompositions may also contain PEG-4000 and/or palmitic acid. Any of thegases disclosed herein or known to the skilled artisan may be employed;however, inert gases, such as SF₆, or fluorocarbons, such as CF₄, C₃F₈and C₄F₁₀, are preferred.

The preferred microbubble suspensions may be prepared from phospholipidsusing known processes such as a freeze-drying or spray-drying solutionsof the crude phospholipids in a suitable solvent or using the processesset forth in EP 554213, U.S. Pat. No. 5,413,774, U.S. Pat. No.5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610, U.S. Pat. No.5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat. No. 5,271,928,U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531,980, U.S. Pat. No.5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat. No. 5,643,553, U.S. Pat.No. 5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No. 6,136,293, EP619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No.5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No. 5,908,610, each ofwhich is incorporated by reference herein in its entirety. Mostpreferably, the phospholipids are dissolved in an organic solvent andthe solution is dried without going through a liposome formation stage.This can be done by dissolving the phospholipids in a suitable organicsolvent together with a hydrophilic stabilizer substance or a compoundsoluble both in the organic solvent and water and freeze-drying orspray-drying the solution. In this embodiment the criteria used forselection of the hydrophilic stabilizer is its solubility in the organicsolvent of choice. Examples of hydrophilic stabilizer compounds solublein water and the organic solvent are e.g. a polymer, like polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG),etc., malic acid, glycolic acid, maltol and the like. Such hydrophiliccompounds also aid in homogenizing the microbubbles size distributionand enhance stability under storage. Any suitable organic solvent may beused as long as its boiling point is sufficiently low and its meltingpoint is sufficiently high to facilitate subsequent drying. Typicalorganic solvents include, for example, dioxane, cyclohexanol, tertiarybutanol, tetrachlorodifluoro ethylene (C₂Cl₄F₂) or 2-methyl-2-butanolhowever, 2-methyl-2-butanol and C₂Cl₄F₂ are preferred.

Prior to formation of the suspension of microbubbles by dispersion in anaqueous carrier, the freeze-dried or spray-dried phospholipid powdersare contacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments that will stabilize themicrobubbles of the gas dispersed therein. This method permitsproduction of suspensions of microbubbles that are stable even whenstored for prolonged periods and are obtained by simple dissolution ofthe dried laminarized phospholipids (which have been stored under adesired gas) without shaking or any violent agitation.

Alternatively, microbubbles can be prepared by suspending a gas into anaqueous solution at high agitation speed, as disclosed e.g. in WO97/29783. A further process for preparing microbubbles is disclosed inco-pending European patent application no. 03002373, herein incorporatedby reference, which comprises preparing an emulsion of an organicsolvent in an aqueous medium in the presence of a phospholipid andsubsequently lyophilizing said emulsion, after optional washing and/orfiltration steps.

Additives known to those of ordinary skill in the art can be included inthe suspensions of stabilized microbubbles. For instance, non-filmforming surfactants, including polyoxypropylene glycol andpolyoxyethylene glycol and similar compounds, as well as variouscopolymers thereof; fatty acids such as myristic acid, palmitic acid,stearic acid, arachidic acid or their derivatives, ergosterol,phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate,ascorbyl palmitate and butylated hydroxytoluene may be added. The amountof these non-film forming surfactants is usually up to 50% by weight ofthe total amount of surfactants but preferably between 0 and 30% byweight.

Other gas containing suspensions include those disclosed in, forexample, U.S. Pat. No. 5,798,091 and WO 97/29783, incorporated herein byreference in their entirety. These agents may be prepared as describedin U.S. Pat. No. 5,798,091 or WO97/29783, each of which is incorporatedby reference in its entirety.

Another preferred ultrasound contrast agent comprises microballoons. Theterm “microballoon” refers to gas filled bodies with a material boundaryor envelope. More on microballoon formulations and methods ofpreparation may be found in EP-A-0 324 938 U.S. Pat. No. 4,844,882; U.S.Pat. No. 5,711,933; U.S. Pat. No. 5,840,275; U.S. Pat. No. 5,863,520;U.S. Pat. No. 6,123,922; U.S. Pat. No. 6,200,548; U.S. Pat. No.4,900,540; U.S. Pat. No. 5,123,414; U.S. Pat. Nos. 5,230,882; 5,469,854;5,585,112; U.S. Pat. No. 4,718,433; U.S. Pat. No. 4,774,958; WO 9501187;U.S. Pat. No. 5,529,766; U.S. Pat. No. 5,536,490 and U.S. Pat. No.5,990,263, each of which is incorporated herein by reference in itsentirety.

The preferred microballoons have an envelope including a biodegradablephysiologically compatible polymer or, a biodegradable solid lipid. Thepolymers useful for the preparation of the microballoons of the presentinvention can be selected from the biodegradable physiologicallycompatible polymers, such as any of those described in any of thefollowing patents: EP 458745, U.S. Pat. No. 5,711,933, U.S. Pat. No.5,840,275, EP 554213, U.S. Pat. No. 5,413,774 and U.S. Pat. No.5,578,292, the entire contents of which are incorporated herein byreference. In particular, the polymer can be selected from biodegradablephysiologically compatible polymers, such as polysaccharides of lowwater solubility, polylactides and polyglycolides and their copolymers,copolymers of lactides and lactones such as ε-caprolactone,γ-valerolactone and polypeptides. Other suitable polymers includepoly(ortho)esters (see e.g., U.S. Pat. No. 4,093,709; U.S. Pat. No.4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646); polylacticand polyglycolic acid and their copolymers, for instance DEXON (see J.Heller, Biomaterials 1 (1980), 51; poly(DL-lactide-co-ε-caprolactone),poly(DL-lactide-co-γ-valerolactone),poly(DL-lactide-co-γ-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones (A.S. Angeloni, P. Ferruti, M. Tramontini and M. Casolaro, The Mannichbases in polymer synthesis: 3. Reduction of poly(beta-aminoketone)s topoly(gamma-aminoalcohol)s and their N-alkylation topoly(gamma-hydroxyquaternary ammonium salt)s, Polymer 23, pp 1693-1697,1982.); polyphosphazenes (Allcock, Harry R. Polyphosphazenes: newpolymers with inorganic backbone atoms (Science 193(4259), 1214-19(1976)) and polyanhydrides. The microballoons of the present inventioncan also be prepared according to the methods of WO-A-96/15815,incorporated herein by reference, where the microballoons are made froma biodegradable membrane comprising biodegradable lipids, preferablyselected from mono- di-, tri-glycerides, fatty acids, sterols, waxes andmixtures thereof. Preferred lipids are di- or tri-glycerides, e.g. di-or tri-myristin, -palmityn or -stearin, in particular tripalmitin ortristearin.

The microballoons may employ any of the gases disclosed herein or knownto the skilled artisan; however, inert gases such as fluorinated gasesare preferred. The microballoons may be suspended in a pharmaceuticallyacceptable liquid carrier with optional additives known to those ofordinary skill in the art and stabilizers.

Other gas-containing contrast agent formulations include microparticles(especially aggregates of microparticles) having gas contained thereinor otherwise associated therewith (for example being adsorbed on thesurface thereof and/or contained within voids, cavities or porestherein). Methods for the preparation of these agents are as describedin EP 0122624, EP 0123235, EP 0365467, U.S. Pat. No. 5,558,857, U.S.Pat. No. 5,607,661, U.S. Pat. No. 5,637,289, U.S. Pat. No. 5,558,856,U.S. Pat. No. 5,137,928, WO 9521631 and WO 9313809, each of which isincorporated herein by reference in its entirety.

Any of these ultrasound compositions should also be, as far as possible,isotonic with blood. Hence, before injection, small amounts of isotonicagents may be added to any of above ultrasound contrast agentsuspensions. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2.6% glycerol solution, 5% dextrose solution, etc. Additionally, theultrasound compositions may include standard pharmaceutically acceptableadditives, including, for example, emulsifying agents, viscositymodifiers, cryoprotectants, lyoprotectants, bulking agents etc.

Any biocompatible gas may be used in the ultrasound contrast agentsuseful in the invention. The term “gas” as used herein includes anysubstances (including mixtures) substantially in gaseous form at thenormal human body temperature. The gas may thus include, for example,air; nitrogen; oxygen; CO₂; argon; xenon or krypton, fluorinated gases(including for example, perfluorocarbons, SF₆, SeF₆) a low molecularweight hydrocarbon (e.g. containing from 1 to 7 carbon atoms), forexample, an alkane such as methane, ethane, a propane, a butane or apentane, a cycloalkane such as cyclopropane, cyclobutane orcyclopentene, an alkene such as ethylene, propene, propadiene or abutene, or an alkyne such as acetylene or propyne and/or mixturesthereof. However, fluorinated gases are preferred. Fluorinated gasesinclude materials which contain at least one fluorine atom such as SF₆,freons (organic compounds containing one or more carbon atoms andfluorine, i.e. CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀, CBrF ₃, CCI₂F₂, C₂CIF₅, andCBrClF₂) and perfluorocarbons. The term perfluorocarbon refers tocompounds containing only carbon and fluorine atoms and includes, inparticular, saturated, unsaturated, and cyclic perfluorocarbons. Thesaturated perfluorocarbons, which are usually preferred, have theformula C_(n)F_(n+2), where n is from 1 to 12, preferably from 2 to 10,most preferably from 3 to 8 and even more preferably from 3 to 6.Suitable perfluorocarbons include, for example, CF₄, C₂F₆, C₃F₈ C₄F₈,C₄F₁₀, C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, and C₉F₂₀. Most preferably the gas orgas mixture comprises SF₆ or a perfluorocarbon selected from the groupconsisting of C₃F₈ C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, with C₄F₁₀being particularly preferred. See also WO 97/29783, WO 98/53857, WO98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO98/05364, and WO 98/17324.

In certain circumstances it may be desirable to include a precursor to agaseous substance (e.g. a material that is capable of being converted toa gas in vivo, often referred to as a “gas precursor”). Preferably thegas precursor and the gas it produces are physiologically acceptable.The gas precursor may be pH-activated, photo-activated, temperatureactivated, etc. For example, certain perfluorocarbons may be used astemperature activated gas precursors. These perfluorocarbons, such asperfluoropentane, have a liquid/gas phase transition temperature aboveroom temperature (or the temperature at which the agents are producedand/or stored) but below body temperature; thus, they undergo a phaseshift and are converted to a gas within the human body.

As discussed, the gas can include a mixture of gases. The followingcombinations are particularly preferred gas mixtures: a mixture of gases(A) and (B) in which, at least one of the gases (B), present in anamount of between 0.5-41% by vol., has a molecular weight greater than80 daltons and is a fluorinated gas and (A) is selected from the groupconsisting of air, oxygen, nitrogen, carbon dioxide and mixturesthereof, the balance of the mixture being gas A.

Since ultrasound vesicles may be larger than the other detectable labelsdescribed herein, they may be linked or conjugated to a plurality ofheteromultimeric constructs in order to increase the targetingefficiency of the agent. Attachment to the ultrasound contrast agentsdescribed above (or known to those skilled in the art) may be via directcovalent bond between a binding polypeptide and the material used tomake the vesicle or via a linker, as described previously. For example,see WO 98/53857 generally for a description of the attachment of apeptide to a bifunctional PEG linker, which is then reacted with aliposome composition. See also, Lanza et al., Ultrasound in Med. & Bio.,23(6): 863-870 (1997).

A number of methods may be used to prepare suspensions of microbubblesconjugated to heteromultimers. For example, one may preparemaleimide-derivatized microbubbles by incorporating 5% (w/w) of N-MPB-PE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido-phenylbutyramide), (Avanti Polar-Lipids, Inc) in the phospholipid formulation.Then, solutions of mercaptoacetylated heteromultimers (10 mg/mL in DMF),which have been incubated in deacetylation solution (50 mM sodiumphosphate, 25 mM EDTA, 0.5 M hydroxylamine HCl, pH 7.5) are added to themaleimide-activated microbubble suspension. After incubation in thedark, under gentle agitation, the heteromultimer conjugated microbubblesmay be purified by centrifugation.

Compounds that can be used for derivatization of microbubbles typicallyinclude the following components: (a) a hydrophobic portion, compatiblewith the material forming the envelope of the microbubble or of themicroballoon, in order to allow an effective incorporation of thecompound in the envelope of the vesicel; said portion is representedtypically by a lipid moiety (dipalmitin, distearoyl); and (b) a spacer(typically PEGs of different molecular weights), which may be optionalin some cases (microbubbles may for instance present difficulties to befreeze dried if the spacer is too long e.g) or preferred in some others(e.g. peptides may be less active when conjugated to a microballoon withshort spacers); and (c) a reactive group capable of reacting with acorresponding reacting moiety on the peptide to be conjugated (e.g.maleimido with the —SH group of cysteine).

Alternatively, heteromultimers conjugated to microbubbles may beprepared using biotin/avidin. For example, avidin-conjugatedmicrobubbles may be prepared using a maleimide-activated phospholipidmicrobubble suspension, prepared as described above, which is added tomercaptoacetylated-avidin (which has been incubated with deacetylationsolution). Biotinylated heteromultimers (prepared as described herein),are then added to the suspension of avidin-conjugated microbubbles,yielding a suspension of microbubbles conjugated to the heteromultimers.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilized residue may be stored andtransported without need of temperature control of its environment andin particular it may be supplied to hospitals and physicians for on siteformulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities. Preferably insuch a case it can be supplied in the form of a two-component kit, whichcan include two separate containers or a dual-chamber container. In theformer case preferably the container is a conventional septum-sealedvial, wherein the vial containing the lyophilized residue of step b) issealed with a septum through which the carrier liquid may be injectedusing an optionally prefilled syringe. In such a case the syringe usedas the container of the second component is also used then for injectingthe contrast agent. In the latter case, preferably the dual-chambercontainer is a dual-chamber syringe and once the lyophilizate has beenreconstituted and then suitably mixed or gently shaken, the containercan be used directly for injecting the contrast agent. In both casesmeans for directing or permitting application of sufficient bubbleforming energy into the contents of the container are provided. However,as noted above, in the stabilised contrast agents according to theinvention the size of the gas microbubbles is substantially independentof the amount of agitation energy applied to the reconstituted driedproduct. Accordingly, no more than gentle hand shaking is generallyrequired to give reproducible products with consistent microbubble size.

It can be appreciated by one ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g. a targeting ligand to belinked to the phospholipid during reconstitution), it can be packagedwith the other components of the kit, preferably in a form or containeradapted to facilitate ready combination with the other components of thekit.

No specific containers, vial or connection systems are required; thepresent invention may use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirable substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.As to the stopper, it may be a compound or multicomponent formulationbased on an elastomer, such as poly(isobutylene) or butyl rubber.

Ultrasound imaging techniques which may be used in accordance with thepresent invention include known techniques, such as color Doppler, powerDoppler, Doppler amplitude, stimulated acoustic imaging, and two- orthree-dimensional imaging techniques. Imaging may be done in harmonic(resonant frequency) or fundamental modes, with the second harmonicpreferred.

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles may, for example, be administered in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.Microballoons-containing contrast agents are typically administered indoses such that the amount of wall-forming polymer or lipid is fromabout 10 μg/kg to about 20 mg/kg of body weight.

In a preferred embodiment, the ultrasound contrast agents describedherein are conjugated to one or more heteromultimers comprised of KDR orVEGF/KDR complex binding moieties, and target tissue expressing KDR.These targeted ultrasound contrast agents will localize at sites ofangiogenesis and other tissue expressing KDR and may demonstratesuperior imaging of angiogenic tissue. In another preferred embodiment,the ultrasound contrast agents described herein are conjugated to one ormore heteromultimers comprised of or cMet or HGF/cMet complex bindingmoieties, and target tissue expressing cMet. These targeted ultrasoundcontrast agents will localize at sites of hyperproliferation orangiogenesis (including tumors) and other tissue expressing cMet and maydemonstrate superior imaging of such tissue.

C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging

In accordance with the present invention, a number of optical parametersmay be employed to determine the location of a target, such as a KDR,VEGF/KDR complex, cMet or HGF/cMet complex, with in vivo light imagingafter injection of the subject with an optically-labeledheteromultimeric construct. Optical parameters to be detected in thepreparation of an image may include transmitted radiation, absorption,fluorescent or phosphorescent emission, light reflection, changes inabsorbance amplitude or maxima, and elastically scattered radiation. Forexample, biological tissue is relatively translucent to light in thenear infrared (NIR) wavelength range of 650-1000 nm. NIR radiation canpenetrate tissue up to several centimeters, permitting the use ofheteromultimeric contructs of the invention to image target-containingtissue in vivo. For example, heteromultimeric constructs comprised ofKDR, VEGF/KDR complex, cMet, or HGF/cMet binding polypeptides may beused for optical imaging of KDR, VEGF/KDR complex, cMet, or HGF/cMetcomplex in vivo.

In another embodiment, the heteromultimeric constructs of the inventionmay be conjugated with photolabels, such as optical dyes, includingorganic chromophores or fluorophores, having extensive delocalized ringsystems and having absorption or emission maxima in the range of400-1500 nm. The compounds of the invention may alternatively bederivatized with a bioluminescent molecule. The preferred range ofabsorption maxima for photolabels is between 600 and 1000 nm to minimizeinterference with the signal from hemoglobin. Preferably,photoabsorption labels have large molar absorptivities, e.g. >10⁵cm⁻¹M⁻¹, while fluorescent optical dyes will have high quantum yields.Examples of optical dyes include, but are not limited to those describedin WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and references citedtherein. For example, the photolabels may be covalently linked directlyto heteromultimers of the invention, such as, for example,heteromultimers comprised of KDR or VEGF/KDR complex binding peptides orlinked to such a heteromultimers via a linker, as described previously.

After injection of the optically-labeled heteromultimeric construct, thepatient is scanned with one or more light sources (e.g., a laser) in thewavelength range appropriate for the photolabel employed in the agent.The light used may be monochromatic or polychromatic and continuous orpulsed. Transmitted, scattered, or reflected light is detected via aphotodetector tuned to one or multiple wavelengths to determine thelocation of target-containing tissue (, e.g., tissue containing KDR,VEGF/KDR complex, cMet, or HGF/cMet complex) in the subject. Changes inthe optical parameter may be monitored over time to detect accumulationof the optically-labeled reagent at the target site (e.g. the site ofangiogenesis). Standard image processing and detecting devices may beused in conjunction with the optical imaging reagents of the presentinvention.

The optical imaging reagents described above may also be used foracousto-optical or sonoluminescent imaging performed withoptically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO98/57666, and references therein). In acousto-optical imaging,ultrasound radiation is applied to the subject and affects the opticalparameters of the transmitted, emitted, or reflected light. Insonoluminescent imaging, the applied ultrasound actually generates thelight detected. Suitable imaging methods using such techniques aredescribed in WO 98/57666.

D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy.

Heteromultimers of the invention may be conjugated with a radionuclidereporter appropriate for scintigraphy, SPECT or PET imaging or with aradionuclide appropriate for radiotherapy. Constructs in which theheteromultimers of the invention are conjugated with both a chelator fora radionuclide useful for diagnostic imaging and a chelator for aradionuclide useful for radiotherapy are within the scope of theinvention.

For use as a PET agent, a heteromultimer may be complexed with one ofthe various positron emitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu,⁶⁸Ga, ⁷²As, ^(94m)Tc, or ¹¹⁰In. The heteromultimeric constructs can alsobe labeled by halogenation using radionuclides, such as ¹⁸F, ¹²⁴I, ¹²⁵I,¹³¹I, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclides for scintigraphy orradiotherapy include ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm,¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy,⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi,²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pb, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au.The choice of metal or halogen will be determined based on the desiredtherapeutic or diagnostic application. For example, for diagnosticpurposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,and ¹¹¹In. For therapeutic purposes, the preferred radionuclides include⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁷⁵Yb, ¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au. ^(99m)Tc is particularlypreferred for diagnostic applications because of its low cost,availability, imaging properties, and high specific activity. Thenuclear and radioactive properties of Tc-99m make this isotope an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹Mo-^(99m)Tc generator.

The metal radionuclides may be chelated by, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S.Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556,U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelatorsknown in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA,TETA, and bisamino bisthiol (BAT) chelators (see also U.S. Pat. No.5,720,934). For example, N₄ chelators are described in U.S. Pat. Nos.6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487.Certain N₃S chelators are described in PCT/CA94/00395, PCT/CA94/00479,PCT/CA95/00249 and in U.S. Pat. Nos. 5,662,885; 5,976,495; and5,780,006. The chelator may also include derivatives of the chelatingligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains anN₃S, and N₂S₂ systems such as MAMA (monoamidemonoaminedithiols), DADS(N₂S diaminedithiols), CODADS and the like. These ligand systems and avariety of others are described in Liu and Edwards, Chem. Rev. 1999, 99,2235-2268 and references therein.

The chelator may also include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. Nos. 5,183,653; 5,387,409; and 5,118,797, thedisclosures of which are incorporated by reference herein, in theirentirety.

In another embodiment, disulfide bonds of a binding polypeptide of theinvention are used as two ligands for chelation of a radionuclide suchas ^(99m)Tc. In this way the peptide loop is expanded by theintroduction of Tc (peptide-S—S-peptide changed topeptide-S—Tc—S-peptide). This has also been used in other disulfidecontaining peptides in the literature (J. Q. Chen, A. Cheng, N. K. Owen,T. H. Hoffman, Y. Miao, S. S. Jurisson, T. P. Quinn. J. Nucl. Med. 2001,42, 1847-1855) while maintaining biological activity. The otherchelating groups for Tc can be supplied by amide nitrogens of thebackbone, another cystine amino acid or other modifications of aminoacids.

Particularly preferred metal chelators include those of Formula 20, 21,and 22 (for ¹¹¹In and lanthanides such as paramagnetic Gd³⁺ andradioactive lanthanides, such as, for example ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, and¹⁶⁶Ho) and those of Formula 23, 24, and 25 (for radioactive ^(99m)Tc,¹⁸⁶Re, and ¹⁸⁸Re) set forth below. These and other metal chelatinggroups are described in U.S. Pat. Nos. 6,093,382 and 5,608,110, whichare incorporated by reference herein in their entirety. Additionally,the chelating group of formula 22 is described in, for example, U.S.Pat. No. 6,143,274; the chelating group of formula 24 is described in,for example, U.S. Pat. Nos. 5,627,286 and 6,093,382, and the chelatinggroup of formula 25 is described in, for example, U.S. Pat. Nos.5,662,885; 5,780,006; and 5,976,495.

In the above Formulas 20 and 21, R is alkyl, preferably methyl. In theabove Formula 24, X is either CH₂ or O, Y is either C₁-C₁₀ branched orunbranched alkyl; Y is aryl, aryloxy, arylamino, arylaminoacyl; Y isarylkyl—where the alkyl group or groups attached to the aryl group areC₁-C₁₀ branched or unbranched alkyl groups, C₁-C₁₀ branched orunbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups, J is C(═O)—, OC(═O)—, SO₂—, NC(═O)—,NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—, homopolyamides orheteropolyamines derived from synthetic or naturally occurring aminoacids; all where n is 1-100. Other variants of these structures aredescribed, for example, in U.S. Pat. No. 6,093,382. The disclosures ofeach of the foregoing patents, applications and references areincorporated by reference herein, in their entirety.

The chelators may be covalently linked directly to the heteromultimersor linked to heteromultimers via a linker, as described previously, andthen directly labeled with the radioactive metal of choice (see, WO98/52618, U.S. Pat. No. 5,879,658, and U.S. Pat. No. 5,849,261).

Complexes of radioactive technetium are particularly useful fordiagnostic imaging and complexes of radioactive rhenium are particularlyuseful for radiotherapy. In forming a complex of radioactive technetiumwith the reagents of this invention, the technetium complex, preferablya salt of Tc-99m pertechnetate, is reacted with the reagent in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions; the most preferred reducing agent is stannouschloride. Means for preparing such complexes are conveniently providedin a kit form comprising a sealed vial containing a predeterminedquantity of a reagent of the invention to be labeled and a sufficientamount of reducing agent to label the reagent with Tc-99m.Alternatively, the complex may be formed by reacting a heteromultimer ofthis invention conjugated with an appropriate chelator with a pre-formedlabile complex of technetium and another compound known as a transferligand. This process is known as ligand exchange and is well known tothose skilled in the art. The labile complex may be formed using suchtransfer ligands as tartrate, citrate, gluconate or mannitol, forexample. Among the Tc-99m pertechnetate salts useful with the presentinvention are included the alkali metal salts such as the sodium salt,or ammonium salts or lower alkyl ammonium salts. Preparation of theComplexes of the Present Invention where the Metal is Radioactiverhenium may be accomplished using rhenium starting materials in the +5or +7 oxidation state. Examples of compounds in which rhenium is in theRe(VII) state are NH₄ReO₄ or KReO₄. Re(V) is available as, for example,[ReOCl₄](NBu₄), [ReOCl₄](AsPh₄), ReOCl₃(PPh₃)₂ and as ReO₂(pyridine)₄ ⁺.(Ph is phenyl; Bu is n-butyl). Other rhenium reagents capable of forminga rhenium complex may also be used.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) to100 mCi per mL.

Generally, the unit dose to be administered has a radioactivity of about0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution tobe injected at unit dosage is from about 0.01 mL to about 10 mL.

Typical doses of a radionuclide-labeled heteromultimeric constructimaging agent of the invention provide 10-50 mCi. After injection of theheteromultimeric radionuclide imaging agent into the patient, a PETcamera or a gamma camera calibrated for the gamma ray energy of thenuclide incorporated in the imaging agent is used to image areas ofuptake of the agent and quantify the amount of radioactivity present inthe site. Imaging of the site in vivo can take place in a matter of afew minutes. However, imaging can take place, if desired, in hours oreven longer, after the radiolabeled peptide is injected into a patient.In most instances, a sufficient amount of the administered dose willaccumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos.

Proper dose schedules for the radiotherapeutic compounds of the presentinvention are known to those skilled in the art. The compounds can beadministered using many methods which include, but are not limited to, asingle or multiple IV or IP injections, using a quantity ofradioactivity that is sufficient to cause damage or ablation of thetargeted tissue, but not so much that substantive damage is caused tonon-target (normal tissue). The quantity and dose required is differentfor different constructs, depending on the energy and half-life of theisotope used, the degree of uptake and clearance of the agent from thebody and the mass of the tumor. In general, doses can range from asingle dose of about 30-50 mCi to a cumulative dose of up to about 3Curies.

The radiotherapeutic compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and may include, for example, para-aminobenzoic acid, ascorbic acid,gentistic acid and the like.

A single, or multi-vial kit that contains all of the components neededto prepare the radiopharmaceuticals of this invention, other than theradionuclide, is an integral part of this invention.

A single-vial kit preferably contains a chelating ligand (if a metalradionuclide is used), a source of stannous salt (if reduction isrequired, e.g., when using technetium), or other pharmaceuticallyacceptable reducing agent, and is appropriately buffered withpharmaceutically acceptable acid or base to adjust the pH to a value ofabout 3 to about 9. The quantity and type of reducing agent used woulddepend highly on the nature of the exchange complex to be formed. Theproper conditions are well known to those that are skilled in the art.It is preferred that the kit contents be in lyophilized form. Such asingle vial kit may optionally contain labile or exchange ligands suchas glucoheptonate, gluconate, mannitol, malate, citric or tartaric acidand can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or α, β, or γ cyclodextrin that serve to improve theradiochemical purity and stability of the final product. The kit mayalso contain stabilizers, bulking agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial may contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g. the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains theligand, as well as buffers appropriate to adjust the pH to its optimalvalue. After a reaction time of about 5 to 60 minutes, the complexes ofthe present invention are formed. It is advantageous that the contentsof both vials of this multi-vial kit be lyophilized. As above, reactionmodifiers, exchange ligands, stabilizers, bulking agents, etc. may bepresent in either or both vials.

Other Therapeutic Applications

The heteromultimeric constructs of the present invention can be used toimprove the activity and/or efficacy of therapeutic agents by, forexample, improving their affinity for or residence time at the target.In this embodiment heteromultimers are conjugated with the therapeuticagent. Alternatively, as discussed above, a liposome or bubblecontaining a therapeutic agent may be conjugated to heteromultimers ofthe invention. The therapeutic agent may be a radiotherapeutic,discussed above, a drug, chemotherapeutic or tumorcidal agent, geneticmaterial, or a gene delivery vehicle, etc. The heteromultimer portion ofthe conjugate causes the therapeutic to “home” to the sites of targetexpression/localization and to improve the affinity of the conjugate forthese sites, so that the therapeutic activity of the conjugate is morelocalized and concentrated at the target sites. For example, in oneembodiment heteromultimers including KDR or VEGF/KDR complex bindingpolypeptides, can be used to improve the activity of therapeutic agents(such as anti-angiogenic or tumorcidal agents) against undesiredangiogenesis such as occurs in neoplastic tumors, by providing orimproving their affinity for KDR or the VEGF/KDR complex and theirresidence time at a KDR or VEGF/KDR complex on endothelium undergoingangiogenesis. In this aspect of the invention, hybrid agents areprovided by conjugating KDR or VEGF/KDR complex binding heteromultimerswith a therapeutic agent. Such heteromultimeric constructs will beuseful in treating angiogenesis associated diseases, especiallyneoplastic tumor growth and metastasis, in mammals, including humans.The method of treatment comprises administering to a mammal in needthereof an effective amount of a heteromultimeric construct comprisingKDR or VEGF/KDR complex binding polypeptides conjugated with atherapeutic agent. The invention also provides the use of suchconjugates in the manufacture of a medicament for the treatment ofangiogenesis associated diseases in mammals, including humans.Heteromultimeric constructs of the invention comprising cMet or HGF/cMetcomplex binding moieties may be used similarly to treat diseaseassociated with hyperproliferation or angiogenesis.

Suitable therapeutic agents for use in this aspect of the inventioninclude, but are not limited to: antineoplastic agents, such as platinumcompounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside,arabinosyl adenine, mercaptopolylysine, vincristine, busulfan,chlorambucil, melphalan (e.g., PAM, a, L-PAM or phenylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin(actinomycin D), daunorubcin, hydrochloride, doxorubicin hydrochloride,taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, acetate, megestrol acetate,tamoxifen citrate, testolactone, trilostane, amsacrine (m-AMSA),ASPARAGINASE (L-ASPARAGINASE) Erwina aparaginase, etoposide (VP-16),interferon cx-2a, Interferon cx-2b, teniposide (VM-26, vinblastinesulfate (VLB), vincristine sulfate, bleomycin sulfate, adriamycin, andarabinosyl; anti-angiogenic agents such as; tyrosine kinase inhibitorswith activity toward signaling molecules important in angiogenesisand/or tumor growth such as SU5416 and SU6668 (Sugen/Pharmacia &Upjohn), endostatin (EntreMed), angiostatin (EntreMed), Combretastatin(oxigene), cyclosporine, 5-fluorouracil, vinblastine, doxorubicin,paclitaxel, daunorubicin, immunotoxins; coagulation factors; antiviralssuch as acyclovir, amantadine azidothymidine (AZT or Zidovudine),ribavirin and vidarabine monohydrate (adenine arahinoside, ara-A);antibiotics, antimalarials, antiprotozoans such as chloroquine,hydroxychloroquine, metronidazole, quinine and meglumine antimonate;anti-inflammatories such as diflunisal, ibuprofen, indomethacin,meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates.

Where heteromultimeric constructs target other tissue and are useful intreating other disease states the skilled artisan may substitute anappropriate therapeutic agent.

The heteromultimeric constructs of the present invention may also beused to target genetic material to specific cells. For example, theheteromultimeric constructs of the present invention may be used tolocalize genetic material to cells or tissue containing the desiredtarget. Thus such constructs may be useful in gene therapy. The geneticmaterial may include nucleic acids, such as RNA or DNA, of eithernatural or synthetic origin, including recombinant RNA and DNA andantisense RNA and DNA. Types of genetic material that may be usedinclude, for example, genes carried on expression vectors such asplasmids, phagemids, cosmids, yeast artificial chromosomes (YACs) anddefective or “helper” viruses, antigene nucleic acids, both single anddouble stranded RNA and DNA and analogs thereof, such asphosphorothioate and phosphorodithioate oligodeoxynucleotides.Additionally, the genetic material may be combined, for example, withlipids, proteins or other polymers. Delivery vehicles for geneticmaterial may include, for example, a virus particle, a retroviral orother gene therapy vector, a liposome, a complex of lipids (especiallycationic lipids) and genetic material, a complex of dextran derivativesand genetic material, etc.

In a preferred embodiment the heteromultimeric constructs of theinvention are utilized in gene therapy for treatment of diseasesassociated with angiogenesis. In this embodiment, genetic material, orone or more delivery vehicles containing genetic material, e.g., usefulin treating an angiogenesis-related disease, may be conjugated to one ormore KDR or VEGF/KDR complex binding heteromultimers or cMet or HGF/cMETcomplex binding heteromultimers of the invention and administered to apatient.

Constructs including genetic material and the KDR bindingheteromultimers of the invention may be used, in particular, toselectively introduce genes into angiogenic endothelial cells, which maybe useful not only to treat cancer, but also after angioplasty, whereinhibition of angiogenesis may inhibit restenosis.

Therapeutic agents and heteromultimers of the invention can be linked orfused in known ways, using the same type of linkers discussed herein.Preferred linkers will be substituted or unsubstituted alkyl chains,amino acid chains, polyethylene glycol chains, and other simplepolymeric linkers known in the art. More preferably, if the therapeuticagent is itself a protein, for which the encoding DNA sequence is known,the therapeutic protein and a binding polypeptide of the invention maybe coexpressed from the same synthetic gene, created using recombinantDNA techniques, as described above. For example, the coding sequence fora binding polypeptide may be fused in frame with that of the therapeuticprotein, such that the peptide is expressed at the amino- orcarboxy-terminus of the therapeutic protein, or at a place between thetermini, if it is determined that such placement would not destroy therequired biological function of either the therapeutic protein or thebinding polypeptide. A particular advantage of this general approach isthat concatamerization of multiple, tandemly arranged bindingpolypeptides is possible, thereby increasing the number andconcentration of binding sites associated with each therapeutic protein.In this manner binding peptide avidity is increased which would beexpected to improve the efficacy of the recombinant therapeutic fusionprotein.

Similar recombinant proteins containing one or more coding sequences fora binding polypeptide may be useful in imaging or therapeuticapplications. For example, in a variation of the pre-targetingapplications discussed infra, the coding sequence for a KDR, VEGF/KDRcomplex, cMet, or HGF/cMet binding peptide may be fused in frame to asequence encoding an antibody (or an antibody fragment or recombinantDNA construct including an antibody, etc.) which, for example, binds toa chelator for a radionuclide (or another detectable label). Theantibody expressing the KDR, VEGF/KDR complex, cMet, or HGF/cMet bindingpolypeptide is then administered to a patient and allowed to localizeand bind to KDR- or cMet-expressing tissue. After the non-bindingantibodies have been allowed to clear, the chelator-radionuclide complex(or other detectable label), which the antibody recognizes isadministered, permitting imaging of or radiotherapy to the KDR- orcMet-expressing tissues. Additionally, the coding sequence for a bindingpeptide may be fused in frame to a sequence encoding, for example, serumproteins or other proteins that produce biological effects (such asapoptosis, coagulation, internalization, differentiation, cellularstasis, immune system stimulation or suppression, or combinationsthereof). The resulting recombinant proteins are useful in imaging,radiotherapy, and therapies directed against cancer and other diseasesthat involve angiogenesis or diseases associated with the pathogensdiscussed herein.

Additionally, heteromultimers of the present invention may themselves beused as therapeutics to treat a number of diseases. For example, wherebinding of a protein or other molecule (e.g. a growth factor, hormoneetc.) is necessary for or contributes to a disease process and a bindingmoiety inhibits such binding, heteromultimers including such bindingmoieties may be useful as therapeutics. Similarly, where binding of abinding moiety itself inhibits a disease process, heteromultimerscontaining such binding moieties may also be useful as therapeutics.

As binding of VEGF and activation of KDR is necessary for angiogenicactivity, in one embodiment heteromultimers including KDR or VEGF/KDRcomplex binding polypeptides that inhibit the binding or inhibit VEGF toKDR (or otherwise inhibit activation of KDR) may be used asanti-angiogenic agents. Certain heteromultimers of the invention thatinhibit activation of KDR are discussed in the Examples. A particularlypreferred heteromultimer is the heterodimer-containing construct D1(structure shown below in Example 9). Other preferred heterodimerconstructs include D4, D5, and D6 (structures provided in Examples 9 and15 below). These and other heteromultimers may be useful in thetreatment of cancer or other diseases associated with inappropriate orexcessive angiogenesis, such as, for example arthritis andatherosclerotic plaques, trachoma, corneal graft neovascularization,psoriasis, sleroderma, hemangioma and hypertrophic scarring, vascularadhesions, angiofibroma, and ocular diseases, such as diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rebeosis, Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibroma andwound granulation. Other conditions that involve angiogenesis include,for example, solid tumors, tumor metastases and benign tumors. Suchtumors and related disorders are well known in the art and include, forexample, melanoma, central nervous system tumors, neuroendocrine tumors,sarcoma, multiple myeloma as wells as cancer of the breast, lung,prostate, colon, head & neck, and ovaries. Additional tumors and relateddisorders are listed in Table I of U.S. Pat. No. 6,025,331, issued Feb.15, 2000 to Moses, et al., the teachings of which are incorporatedherein by reference. Benign tumors include, for example, hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas.Other relevant diseases or conditions that involve blood vessel growthinclude intestinal adhesions, atherosclerosis, scleroderma, andhypertropic scars, and ulcers. Furthermore, the heteromultimers of thepresent invention can be used to reduce or prevent uterineneovascularization required for embryo implantation, for example, as abirth control agent.

Heteromultimers of this invention can also be useful for treatingvascular permeability events that can result when VEGF binds KDR. Inrenal failure it has been shown that anti-VEGF antibodies can reversedamage and in a similar way the compounds of the invention can reverserenal permeability pathogenesis in, for example, diabetes.

As the interruption of the HGF interaction with the cMet receptor slowstumor progression, in another embodiment, the heteromultimers includecMet or HGF/cMet complex binding polypeptides that inhibit the bindingof cMet to HGF (or otherwise inhibit the activation of cMet) may be usedto treat tumors and other hyperproliferative disorders. Particularheteromultimers that inhibit cMet are discussed in the Examples. Apreferred heteromultimer is D28 (structure shown below in Example 9).

Furthermore, heteromultimers of the present invention may be useful intreating diseases associated with certain pathogens, including, forexample, malaria, HIV, SIV, Simian hemorrhagic fever virus, etc.Sequence homology searches of KDR-binding peptides identified by phagedisplay using the BLAST program at NCBI has identified a number ofhomologous proteins known or expected to be present on the surface ofpathogenic organisms. Homologies were noted between KDR and VEGF/KDRcomplex binding polypeptides and proteins from various malaria strains,HIV, SIV, simian hemorrhagic fever virus, and an enterohemorrhagic E.coli strain. Some of the homologous proteins, such as PfEMP1 and EBL-1,are hypermutable adhesion proteins known to play roles in virulence.These proteins possess multiple binding sites that are capable ofbinding to more than one target molecule on the host's surface. Theirhigh mutation and recombination rates allow them to quickly develop newbinding sites to promote survival and/or invasion. Similarly, proteinssuch as gp120 of HIV (which also has homology to some of the KDR-bindingpeptides disclosed herein) play critical roles in the adhesion ofpathogens to their hosts. Although not reported previously, it ispossible that many of the pathogen proteins with homology to theKDR-binding peptides disclosed herein also bind to KDR. Comparison ofthe pathogen protein sequences with the corresponding peptide sequencesmay suggest changes in the peptide sequence or other modifications thatwill enhance its binding properties. Additionally, heteromultimericconstructs including the KDR-binding peptide sequences disclosed hereinmay have usefulness in blocking infection with the pathogen species thatpossesses the homology. Indeed, a strategy is being employed to blockHIV infection by trying to prevent virus envelope proteins from bindingto their known cellular surface targets such as CD4. Howie S E, et al.,FASEB J 1998 August; 12(11):991-8, “Synthetic peptides representingdiscontinuous CD4 binding epitopes of HIV-1 gp120 that induce T cellapoptosis and block cell death induced by gp120.” Thus, KDR mayrepresent a previously unknown target for a number of pathogens and theheteromultimeric constructs including KDR or VEGF/KDR complex bindingpeptides may be useful in treating the diseases associated with thesepathogens.

In the above treatment methods, the compounds may be administered by anyconvenient route customary for therapeutic agents, for exampleparenterally, enterally or intranasally, and preferably by infusion orbolus injection, or by depot or slow release formulation. In a preferredembodiment, the composition may be formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Otherpharmaceutically acceptable carriers include, but are not limited to,sterile water, saline solution, buffered saline (including buffers likephosphate or acetate), alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,paraffin, etc. Where necessary, the composition may also include asolubilizing agent and a local anaesthetic such as lidocaine to easepain at the site of the injection, preservatives, stabilizers, wettingagents, emulsifiers, salts, lubricants, etc. as long as they do notreact deleteriously with the active compounds. Similarly, thecomposition may comprise conventional excipients, i.e. pharmaceuticallyacceptable organic or inorganic carrier substances suitable forparenteral, enteral or intranasal application which do not deleteriouslyreact with the active compounds. Generally, the ingredients will besupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent in activity units. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade “water for injection” orsaline. Where the composition is to be administered by injection, anampoule of sterile water for injection or saline may be provided so thatthe ingredients may be mixed prior to administration. The quantity ofmaterial administered will depend on the seriousness of the condition.For example, for treatment of anangiogenic condition, e.g., in the caseof neoplastic tumor growth, the position and size of the tumor willaffect the quantity of material to be administered. The precise dose tobe employed and mode of administration must per force in view of thenature of the complaint be decided according to the circumstances by thephysician supervising treatment. In general, dosages of theheteromultimer/therapeutic agent conjugate will follow the dosages thatare routine for the therapeutic agent alone, although the improvedaffinity of a heteromultimer of the invention for its target may allow adecrease in the standard dosage.

Such conjugate pharmaceutical compositions are preferably formulated forparenteral administration, and most preferably for intravenous orintra-arterial administration. Generally, and particularly whenadministration is intravenous or intra-arterial, pharmaceuticalcompositions may be given as a bolus, as two or more doses separated intime, or as a constant or non-linear flow infusion.

The hetermultimers can be administered to an individual over a suitabletime course depending on the nature of the condition and the desiredoutcome. The heteromultimeric constructs can be administeredprophylactically, e.g., before the condition is diagnosed or to anindividual predisposed to a condition. Alternatively, theheteromultimers of the invention can be administered while theindividual exhibits symptoms of the condition or after the symptoms havepassed or otherwise been relieved (such as after removal of a tumor). Inaddition, the heteromultimers of the present invention can beadministered a part of a maintenance regimen, for example to prevent orlessen the recurrence or the symptoms or condition. As described herein,the heteromultimers of the present invention can be administeredsystemically or locally.

As used herein the term “therapeutic” includes at least partialalleviation of symptoms of a given condition. The heteromultimericconstructs of the present invention do not have to produce a completealleviation of symptoms to be useful. For example, treatment of anindividual can result in a decrease in the size of a tumor or diseasedarea, or prevention of an increase in size of the tumor or diseased areaor partial alleviation of other symptoms. Treatment can result inreduction in the number of blood vessels in an area of interest or canprevent an increase in the number of blood vessels in an area ofinterest. Treatment can also prevent or lessen the number or size ofmetastic outgrowths of the main tumor(s).

In one embodiment, symptoms that can be alleviated include physiologicalcharacteristics such as VEGF receptor activity and migration ability ofendothelial cells. The heteromultimers of the present invention caninhibit activity of VEGF receptors, including VEGF-2/KDR, VEGF-1/Flt-1and VEGF-3/Flt-4. Such inhibition can also be detected, for example, bymeasuring the phosphorylation state of the receptor in the presence ofor after treatment with the binding polypeptides or constructs thereof.Based on the teachings provided herein, one of ordinary skill in the artwould know how and be able to administer a suitable dose of bindingpolypeptide or construct thereof as provided herein and measured beforeand after treatment. In another embodiment, the phosphorylation state ofthe relevant receptor, or the migration ability of endothelial in anarea of interest can be measured in samples taken from the individual.The VEGF receptors or endothelial cells can be isolated from the sampleand used in assays described herein.

The dosage of the heteromultimers may depend on the age, sex, health,and weight of the individual, a well as the nature of the condition andoverall treatment regimen. The biological effects of the multimers aredescribed herein. Therefore, based on the biological effects of theheteromultimers provided herein, and the desired outcome of treatment,the referred dosage is determinable by one of ordinary skill in the artthrough route optimization procedures. Typically, the daily regiment isin the range of about 0.1 μg/kg to about 1 mg/kg.

The heteromultimers provided herein can be administered as the soleactive ingredient together with a pharmaceutically acceptable excipient,or can be administered together with other binding polypeptides andconstructs thereof, other therapeutic agents, or combination thereof. Inaddition, the heteromultimers can be conjugated to therapeutic agents,for example, to improve specificity, residence time in the body, ortherapeutic effect. Such other therapeutic agents include, for example,other antiangiogenic compounds, and tumoricidal compounds. Thetherapeutic agent can also include antibodies.

Furthermore, the heteromultimers of the present invention can be used asan endothelial cell homing device. Therefore, the heteromultimericconstructs can be conjugated to nucleic acids encoding, for example, atherapeutic polypeptide, in order to target the nucleic acid toendothelial cells. Once exposed to the nucleic acid, thereby deliveringthe therapeutic peptide to the target cells.

In another embodiment of the invention, the therapeutic agent can beassociated with an ultrasound contrast agent composition, saidultrasound contrast agent including the KDR, VEGF/KDR complex, cMet, orHGF/cMet binding peptides of the invention linked to the materialemployed to form the vesicles (particularly microbubbles ormicroballoons) comprised in the contrast agent, as previously described.For instance, said contrast agent/therapeutic agent association can becarried out as described in U.S. Pat. No. 6,258,378, herein incorporatedby reference. Thus, after administration of the ultrasound contrastagent and the optional imaging of the contrast agent bound to thepathogenic site expressing the KDR, VEGF/KDR complex, cMet, or HGF/cMetcomplex, the pathogenic site can be irradiated with an energy beam(preferably ultrasonic, e.g. with a frequency of from 0.3 to 3 MHz), tocause the bursting of microvesicles, as disclosed for instance in theabove cited U.S. Pat. No. 6,258,378. The therapeutic effect of thetherapeutic agent can thus be advantageously enhanced by the energyreleased by the burst of the microvesicles, in particular causing aneffective delivery of the therapeutic agent to the targeted pathogenicsite.

As discussed above, the heteromultimers can be administered by anysuitable route. Suitable routes of administration include, but are notlimited to, topical application, transdermal, parenteral,gastrointestinal, intravaginal, and transalvcolar. Compositions for thedesired route of administration can be prepared by any of the methodswell known in the pharmaceutical arts. Details concerning dosages,dosage forms, modes of administration, composition and the like arefurther discussed in a standard pharmaceutical text, such as Remington'sPharmaceutical Sciences, 18th ed., Alfonso R. Gennaro, ed. (MackPublishing Co., Easton, Pa. 1990), which is hereby incorporated byreference.

For topical applications, the heteromultimers can be suspended, forexample, in a cream, gel or rinse which allows the polypeptides orconstructs to penetrate the skin and enter the blood stream, forsystemic delivery, or contact the are of interest, for localizeddelivery. Compositions suitable for topical application include anypharmaceutically acceptable base in which the polypeptides are at leastminimally soluble.

For transdermal administration, the heteromultimers can be applied inpharmaceutically acceptable suspension together with a suitabletransdermal device or “patch.” Examples of suitable transdermal devicesfor administration of the heteromultimers of the present invention aredescribed, for example, in U.S. Pat. No. 6,165,458, issued Dec. 26, 2000to Foldvari, et al., and U.S. Pat. No. 6,274,166B1, issued Aug. 4, 2001to Sintov, et al., the teachings of which are incorporated herein byreference.

For parenteral administration, the heteromultimers can be suspended, forexample, in a pharmaceutically acceptable sterile isotonic solution,such as saline and phosphate buffered saline. The constructs of theinvention can then be injected intravenously, intramuscularly,intraperitoneally, or subcutaneously.

For gastrointestinal and intravaginal administration, theheteromultimers can be incorporated into pharmaceutically acceptablepowders, pills or liquids for ingestion, and suppositories for rectal orvaginal administration.

For transalveolar, buccal or pulmonary administration, theheteromultimers can be suspended in a pharmaceutically acceptableexcipient suitable for aerosolization and inhalation or as a mouthwash.Devices suitable for transalveolar administration such as atomizers andvaporizes are also included within the scope of the invention. Suitableformulations for aerosol delivery of polypeptides using buccal orpulmonary routes can be found, for example in U.S. Pat. No. 6,312,665B1,issued Nov. 6, 2001 to Pankaj Modi, the teachings of which areincorporated herein by reference.

In addition, the heteromultimers of the present invention can beadministered nasally or ocularly, where the heteromultimers aresuspended in a liquid pharmaceutically acceptable agent suitable fordropwise dosing.

The heteromultimers of the present invention can be administered suchthat the polypeptide is released in the individual over an extendedperiod of time (sustained or controlled release). For example, theheteromultimers can be formulated into a composition such that a singleadministration provides delivery of the constructs of the invention forat least one week, or over the period of a year or more. Controlledrelease systems include monolithic or reservoir-type microcapsules,depot implants, osmotic pumps, vesicles, micelles, liposomes,transdermal patches and iontophoretic devices. In one embodiment, theheteromultimers of the present invention are encapsulated or admixed ina slow degrading, non-toxic polymer. Additional formulations suitablefor controlled release of constructs of the invention are described inU.S. Pat. No. 4,391,797, issued Jul. 5, 1983, to Folkman, et al., theteachings of which are incorporated herein by reference.

Another suitable method for delivering the heteromultimers of thepresent invention to an individual is via in vivo production of thepolypeptides. Genes encoding the polypeptides can be administered to theindividual such that the encoded polypeptides are expressed. The genescan be transiently expressed. In a particular embodiment, the genesencoding the polypeptide are transfected into cells that have beenobtained from the patient, a method referred to as ex vivo gene therapy.Cells expressing the polypeptides are then returned to the patient'sbody. Methods of ex vivo gene therapy are well known in the art, and aredescribed, for example, in U.S. Pat. No. 4,391,797, issued Mar. 21, 1998to Anderson, et al., the teachings of which are incorporated herein byreference.

Preparation and tests of heteromultimeric constructs in accordance withthis invention will be further illustrated in the following examples.The specific parameters included in the following examples are intendedto illustrate the practice of the invention, and they are not presentedto in any way limit the scope of the invention.

Example 1 Peptide Synthesis and Fluorescein Labelling

Selected KDR or VEGF/KDR binding peptides corresponding to positivephage isolates were synthesized on solid phase using9-fluorenylmethoxycarbonyl protocols and purified by reverse phasechromatography. Peptide masses were confirmed by electrospray massspectrometry, and peptides were quantified by absorbance at 280 nm. Forsynthesis, two N-terminal and two C-terminal amino acids from the phagevector sequence from which the peptide was excised were retained and a-Gly-Gly-Gly-Lys-NH₂ linker was added to the C-terminus of each peptide.Peptides with selected lysine residues were protected with1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),which allows selective coupling to the C-terminal lysine, is not removedduring peptide cleavage, and can be removed after coupling with 2%hydrazine in DMF or 0.5 M hydroxylamine, pH 8, in water.

Each peptide was labeled with fluorescein on the C-terminal lysine usingFluorescein (N-hydroxysuccinimide ester derivative) or FluoresceinIsothiocyanate (FITC) in DMF, 2% diisopropylethylamine (DIPEA). If thepeptide contained an ivDde protected lysine, the reaction was quenchedby the addition of 2% hydrazine, which reacts with all freeNHS-fluorescein and removes the internal protecting group. For all otherpeptides, the reaction was quenched by the addition of an equal volumeof 0.5 M hydroxylamine, pH 8. The quenched reactions were then dilutedwith water to less than 10% DMF and then purified using C18 reversephase chromatography. The peptides were characterized for purity andcorrect mass on an LC-MS system (HP1100 HPLC with in-line SCIEX AP150single quadrapole mass spectrometer).

Fluorescence Anisotropy Measurements and BiaCore Assays

Fluorescence anisotropy measurements were performed in 384-wellmicroplates in a volume of 10 μL in binding buffer (PBS, 0.01% Tween-20,pH 7.5) using a Tecan Polarion fluorescence polarization plate reader.In some cases, heparin (0.5 μg/mL) or 10% human serum was added to thebinding buffer. The concentration of fluorescein labeled peptide washeld constant (20 nM) and the concentration of KDR-Fc (or similartarget) was varied. Binding mixtures were equilibrated for 10 minutes inthe microplate at 30° C. before measurement. The observed change inanisotropy was fit to Equation (1) below via nonlinear regression toobtain the apparent K_(D). Equation (1) assumes that the syntheticpeptide and HSA form a reversible complex in solution with 1:1stoichiometry:

$\begin{matrix}{{r_{obs} = {r_{free} + {\left( {r_{bound} - r_{free}} \right)\frac{\left( {K_{D} + {KDR} + P} \right) - \sqrt{\left( {K_{D} + {KDR} + P} \right)^{2} - {4 \cdot {KDR} \cdot P}}}{2 \cdot P}}}},} & (1)\end{matrix}$

where r_(obs) is the observed anisotropy, r_(free) is the anisotropy ofthe free peptide, r_(bound) is the anisotropy of the bound peptide,K_(D) is the apparent dissociation constant, KDR is the total KDRconcentration, and P is the total fluorescein-labeled peptideconcentration.

KDR-Fc (or another protein target) was cross-linked to the dextransurface of a CM5 sensor chip by the standard amine coupling procedure(0.5 mg/mL solutions diluted 1:20 with 50 mM acetate, pH 6.0, R_(L)KDR-Fc=12859). Experiments were performed in HBS-P buffer (0.01 M HEPES,pH 7.4, 0.15 M NaCl, 0.005% polysorbate 20 (v/v)). Peptide solutionsquantitated by extinction coefficient were diluted to 400 nM in HBS-P.Serial dilutions were performed to produce 200, 100, 50, and 25 nMsolutions. For association, peptides were injected at 20 μL/min for 1minute using the kinject program. Following a 1-minute dissociation, anyremaining peptide was stripped from the target surface with a quickinjection of 1M NaCl for 25 sec. at 50 μL/min. All samples were injectedin duplicate. Between each peptide series a buffer injection and anon-target binding peptide injection served as additional controls.Sensorgrams were analyzed using the simultaneous ka/kd fitting programin the BIAevaluation software 3.1.

The following common abbreviations are used throughout thisspecification: 9-fluorenylmethyloxycarbonyl (fmoc or Fmoc),1-hydroxybenozotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC),N-methylpyrrolidinone (NMP), acetic anhydride (Ac₂O),(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivdde),trifluoroacetic acid (TFA), Reagent B(TFA:H₂O:phenol:triisopropylsilane, 88:5:5:2), diisopropylethylamine(DIEA), O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate(HATU), N-hydroxysuccinimide (NHS), solid phase peptide synthesis(SPPS), dimethyl sulfoxide (DMSO), dichloromethane (DCM),dimethylformamide (DMF), human serum albumin (HSA), and radiochemicalpurity (RCP).

Experimental Methods

The following methods were employed in the Examples.

Method 1 for ACT 357 MPS and ACT 496 MOS Synthesizers

The peptides were synthesized on NovaSyn TGR (Rink amide) resin (0.2mmol/g) using the Advanced ChemTech ACT 357 or ACT 496 Synthesizersemploying Fmoc peptide synthesis protocols, specifically using HOBt/DICas the coupling reagents and NMP as the solvent. The Fmoc was removed bytreating the Nova-Syn TGR (Rink amide-available from NovaBiochem, SanDiego, Calif.) resin-bound peptide with 25% piperidine in DMF twice (4min and 10 min). All amino acids were dissolved in NMP (DMF was addedwhen the amino acid was not soluble in pure NMP). The concentration ofthe amino acid was 0.25 M, and the concentration for both HOBt and DICwas 0.5 M.

For a 0.04 mmol scale synthesis:

A typical amino acid coupling cycle (not including wash steps) was todispense piperidine solution (2.4 mL) to each well and mix for 4 min,then empty all wells. NMP (320 μL), HOBt solution (320 μL, 4 eq), aminoacid (640 μL, 4 eq) and DIC (320 μL, 4 eq) solutions were dispensed toeach well. The coupling time was 3 h; then the resin was washed. Thecycle was repeated for each amino acid. After the last amino acidcoupling, the resin-bound peptide was treated with 25% piperidine toremove the Fmoc protecting group. After washing, the resin bound peptidewas capped with 1.0 M Ac₂O (1.2 mL per well) and diisopropylethylaminein DMF, optionally including varying amounts of HOBt in the mixture for30 min. The resin was washed first with methanol and then withdichloromethane and dried. Cleavage of the peptides from the resin andside-chain deprotection was accomplished using Reagent B for 4.5 h. Thecleavage solutions were collected and the resins were washing with anadditional aliquot of Reagant B. The combined solutions wereconcentrated to dryness. Ether was added to the residue with swirling orstirring to precipitate the peptides. The ether was decanted, and solidwas collected. This procedure was repeated 2-3 times to removeimpurities. The crude peptides were dissolved in DMSO and water mixture,and purified by HPLC (column: Water's Associates Xterra C18, 19×50 mm;solvents: H₂O with 0.1% TFA and CH₃CN with 0.1% TFA; UV 220 nm; Flowrate: 50-60 mL/min). The solutions containing the peptide werelyophilized to give the desired peptides as white fluffy lyophilizates(>90% purity).

The purified linear di-cysteine containing peptides were dissolved inwater, mixtures of water-acetonitrile, or mixtures of water-DMSO atconcentrations between 0.1 mg/mL and 2.0 mg/mL. The choice of solventwas a function of the solubility of the crude peptide in the solvent.The pH of the solution was adjusted to 7.5-8.5 with aqueous ammonia,aqueous ammonium carbonate or aqueous ammonium bicarbonate. The mixturewas stirred vigorously in air for 24-48 h. In the case of non-DMSOcontaining solvent systems, the pH of the solution was adjusted to 2with aqueous trifluoroacetic acid. The mixture was lyophilized toprovide the crude cyclic disulfide containing peptide. The cyclicdisulfide peptide was then dissolved to a volume of 1-2 mL in aqueous(0.1% TFA) containing a minimum of acetonitrile (0.1% TFA). Theresulting solution was loaded onto a reverse phase column and thedesired compound obtained by a gradient elution of acetonitrile intowater, employing a C18, or C8 reverse phase semipreparative orpreparative HPLC column. In the case of the DMSO-containing solutions,the solution was diluted until the DMSO concentration was minimalwithout precipitation of the peptide. The resulting mixture was quicklyacidified to pH 2 with dilute trifluoroacetic acid and loaded onto thereverse phase HPLC system and purified as described. Fractionscontaining the desired materials were pooled and the peptides isolatedby lyophilization.

Method 2 for ACT 357 MPS and ACT 496 MOS Synthesizers

The peptides were synthesized as in Method 1, with the followingchanges. HBTU/HOBt/DIEA were used as the coupling reagent and NMP as thesolvent. A low load (˜0.2 mmol/g) Fmoc-GGGK(Boc)-NovSyn-TGR-resinprepared from the above-described Nova-Syn TGR resin was employed forpeptides synthesis on 0.01 mmol scale synthesis.

For a 0.01 mmol scale synthesis:

After the Fmoc group was removed, a standard coupling procedure used asolution of HOBt (720 μL, 6 eq), amino acid (804 μL, 6.6 eq), HBTU (720μL, 6 eq) and DIEA (798 μL, 13.3 eq). The mixture was agitated for 15min, emptied and the resin washed. After all couplings and aftercleavage and purification as above, the solutions containing desiredlinear peptides were lyophilized to give the peptides as white fluffysolids (>90% purity).

The crude ether-precipitated linear di-cysteine containing peptides werecyclized by dissolution in water, mixtures of aqueous acetonitrile (0.1%TFA), or aqueous DMSO and adjustment of the pH of the solution to7.5-8.5 by addition of aqueous ammonia, aqueous ammonium carbonate, oraqueous ammonium bicarbonate solution. The peptide concentration wasbetween 0.1 and 2.0 mg/mL. The mixture was stirred in air for 24-48 h,acidified to a pH of 2 with aqueous trifluoroacetic acid and thenpurified by preparative reverse phase HPLC employing a gradient ofacetonitrile into water. Fractions containing the desired material werepooled and the peptides were isolated by lyophilization.

Method 3 for the ACT 496 MOS Synthesizer

The peptides were synthesized using an Advanced ChemTech ACT 496 MOSSynthesizer as in Method 1. The low load (˜0.2 mmol/g)GGGK(Boc)-NovaSyn-TGR resin was employed for peptide synthesis. Thecoupling solvent was NMP/DMSO 8:2. The synthesis was performed at a 0.02mmol scale using a coupling time of 3 h. The crude linear peptides werefurther processed as described above for Method 1.

Method 4 for the ACT 496 MOS Synthesizer

The peptides were synthesized using method 3 on the ACT 496 withHBTU/DIEA as the coupling reagents, and NMP as the solvent.2,4,6-collidine as a 1 M solution was used as the base. The low loadFmoc-GGGK(ivDde)-Novsyn-TGR resin (˜0.2 mmol/g) was used for peptidesynthesis. The coupling time was 30 minutes. The crude linear peptideswere further processed as described above for Method 1.

Method 5 for the ABI 433A Synthesizer

Synthesis of peptides was carried out on a 0.25 mmol scale using theFastMoc protocol (Applied Biosystems Inc.) In each cycle of thisprotocol, 1.0 mmol of a dry protected amino acid in a cartridge wasdissolved in a solution of 0.9 mmol of HBTU, 2 mmol of DIEA, and 0.9mmol of HOBt in DMF with additional NMP added. The peptides were madeusing 0.1 mmol of NovaSyn TGR (Rink amide) resin (resin substitution 0.2mmol/g). The coupling time in this protocol was 21 min. Fmocdeprotection was carried out with 20% piperidine in NMP. At the end ofthe last cycle, the synthesized peptide was acetylated using aceticanhydride/DIEA/HOBt/NMP. The peptide resin was washed and dried forfurther manipulations or cleaved from the resin (using reagent B).Generally, the cleaved peptides were cyclized, as in Method 1, above.

Method 6 Biotinylation of Resin Bound Peptides

The peptides were prepared by Method 5. The ivDde protecting group onthe C-terminal lysine was selectively removed by treatment with 10%hydrazine in DMF. The resin was then treated with a solution ofBiotin-N-hydroxysuccinimidyl ester in DMF in the presence of DIEA. Afterwashing, the resin was dried and cleavage was performed using Reagent B.The resin was filtered off and the filtrate concentrated to dryness. Thebiotinylated peptide was dissolved in neat DMSO and treated with DIEAand stirred for 4-6 h to effect disulfide cyclization. The crude mixturewas purified by preparative HPLC.

In a typical experiment, 200 mg of the resin-bound peptide was treatedwith 10% hydrazine in DMF (2×20 mL) and washed with DMF (2×20 mL) andthen with dichloromethane (1×20 mL). The resin was resuspended in DMF(10 mL) and treated with a solution of Biotin-NHS ester (0.2 mmol, 5equivalent) and DIEA (0.2 mmol) and the resin was mixed with thereagents for 4 h. The completion of the reaction was checked by theninhydrin test. The peptide was then released from the resin bytreatment with Reagent B (10 mL) for 4 h. The resin was filtered off,Reagent B was removed in vacuo and the peptide was precipitated byaddition of anhydrous ether. The solid formed was collected, washed withether and dried. The solid was dissolved in anhydrous DMSO and themixture was adjusted to pH 7.5 with DIEA and stirred for 4-6 h to effectdisulfide cyclization. The disulfide cyclization reaction was monitoredby analytical HPLC. After completion of the cyclization, the mixturesolution was diluted with 25% acetonitrile in water and directlypurified by HPLC on reverse phase C-18 column using a gradient ofacetonitrile into water (both containing 0.1% TFA). Fractions wereanalyzed by analytical HPLC and those containing the pure product werecollected and lyophilized to obtain the required biotinylated peptide.

Method 7 Biotinylation of Purified Peptides

The purified peptide (10 mg, prepared by methods 1-5) containing a freeamino group was dissolved in anhydrous DMF or DMSO (1 mL) and Biotin-NHSester of (5 equivalents) and DIEA (5 equivalents) were added. Thereaction was monitored by HPLC and after the completion of the reaction(1-2 h), the crude reaction mixture was directly purified by preparativeHPLC. Fractions were analyzed by analytical HPLC and those containingthe pure product were collected and lyophilized to obtain the requiredbiotinylated peptide.

Method 8 Biotinylation of Resin Bound Peptides Containing Linkers

In a typical experiment, 400 mg of the resin-containing peptide (madeusing the ABI-433 A Synthesizer and bearing an ivDde-protected lysine)was treated with 10% hydrazine in DMF (2×20 mL). The resin was washedwith DMF (2×20 mL) and DCM (1×20 mL). The resin was resuspended in DMF(10 mL) and treated with Fmoc-aminodioxaoctanoic acid (0.4 mmol), HOBt(0.4 mmol), DIC (0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. Afterthe reaction, the resin was washed with DMF (2×10 ml) and with DCM (1×10mL). The resin was then treated with 20% piperidine in DMF (2×15 mL) for10 min each time. The resin was washed and the coupling withFmoc-diaminodioxaoctanoic acid and removal of the Fmoc protecting groupwere repeated once more. The resulting resin, containing a peptide witha free amino group, was treated with a solution of Biotin-NHS ester (0.4mmol, 5 equivalent) and DIEA (0.4 mmol, 5 equivalents) in DMF for 2 h.The peptide-resin was washed and dried as described previously and thentreated with reagent B (20 mL) for 4 h. The mixture was filtered and thefiltrate concentrated to dryness. The residue was stirred with ether toproduce a solid that was collected, washed with ether, and dried. Thesolid was dissolved in anhydrous DMSO and the pH adjusted to pH 7.5 withDIEA. The mixture was stirred for 4-6 hr. to effect the disulfidecyclization reaction which was monitored by analytical HPLC. After thecompletion of the cyclization, the DMSO solution was diluted with 25%acetonitrile in water and applied directly to a reverse phase C-18column. Purification was effected using a gradient of acetonitrile intowater (both containing 0.1% TFA). Fractions were analyzed by analyticalHPLC and those containing the pure product were collected andlyophilized to provide the required biotinylated peptide.

Method 9 Formation of 5-Carboxyfluorescein Labeled Peptides

Peptide-resin obtained via from Method 5, containing an ivDde protectinggroup on the epsilon nitrogen of lysine, was mixed with a solution ofhydrazine in DMF (10% hydrazine/DMF, 2×10 mL, 10 min) to remove theivDde group. The epsilon nitrogen of the lysine was labeled withfluorescein-5-isothiocyanate (0.12 mmol) and diisopropylethylamine (0.12mmol) in DMF. The mixture was agitated for 12 h (fluorescein-containingcompounds were protected from light). The resin was then washed with DMF(3×10 mL) and twice with CH₂Cl₂ (10 mL) and dried under nitrogen for 1h. The peptide was cleaved from the resin using Reagent B for 4 h andthe solution collected by filtration. The volatiles were removed underreduced pressure and the residue was dried under vacuum. The peptide wasprecipitated with ether, collected and the precipitate was dried under astream of nitrogen. The precipitate was added to water (1 mg/mL) and thepH of the mixture was adjusted to 8 with 10% aqueous meglumine.Cyclization of the peptide was carried out for 48 h and the solution wasfreeze-dried. The crude cyclic peptide was dissolved in water andpurified by RP-HPLC on a C₁₈ column with linear gradient of acetonitrileinto water (both phases contained 0.1% TFA). Fractions containing thepure product were collected and freeze dried. The peptides werecharacterized by ES-MS and the purity was determined by RP-HPLC (lineargradient of acetonitrile into water/0.1% TFA).

Method 10 Preparation of Peptidic Chelate for Binding to Tc by Couplingof Single Amino Acids

Peptides were synthesized starting with 0.1 mmol of NovaSyn-TGR resin(0.2 mmol/g substitution). Deprotected (ivDde) resin was then treatedaccording to the protocol A for the incorporation of Fmoc (Gly)-OH,Fmoc-Cys(Acm)-OH and, Fmoc-Ser(tBu)-OH.

Protocol A for manual coupling of single amino acid:1. Treat with 4 equivalents of corresponding Fmoc-amino acid and 4.1equivalents of hydroxy benzotriazole and 4.1 equivalents of HOB and 4.1equivalents of DIC for 5 h.2. Wash with DMF (3×10 mL)3. Treat with 20% piperidine in DMF (2×10 mL, 10 min)4. Wash with DMF (3×10 mL)

The Fmoc-protected peptide loaded resin was then treated with 20%piperidine in DMF (2×10 mL, 10 min) and washed with DMF (3×10 mL). Asolution of N,N-dimethylglycine (0.11 mmol), HATU (1 mmol), and DIEA(0.11 mmol) in DMF (10 mL) was then added to the peptide loaded resinand the manual coupling was continued for 5 h. After the reaction theresin was washed with DMF (3×10 mL) and CH₂Cl₂ (3×10 mL) and dried undervacuum.

Method 11 Formation of Mercapto-acetylated Peptides UsingS-Acetylthioglvcolic Acid N-Hydroxysuccinimide Ester

To a solution of a peptide (0.005 mmol, obtained from Methods 1-5 with afree amine) in DMF (0.25 mL) was added S-acetylthioglycolic acidN-hydroxysuccinimide ester (SATA) (0.0055 mmol) and the reaction mixturewas stirred at ambient temperature for 6 h. The volatile were removedunder vacuum and the residue was purified by preparative HPLC usingacetonitrile-water containing 0.1% TFA. Fractions containing the pureproduct were collected and freeze-dried to yield the mercaptoacetylatedpeptide. The mercaptoacetylated peptide was characterized by ESI-MS andthe purity was determined by reverse phase PHLC analysis employing alinear gradient of acetonitrile into water (both containing 0.1% TFA).

Method 12 Formation of Mercaptoacetylated Peptide UsingS-Acetylthioglycolic Acid

Purified peptides from Method 5, after disulfide cyclization, werecoupled with S-acetylthioglycolic acid (1.5-10 eq.)/HOBt (1.5-10eq.)/DIC (1.5-10 eq.) in NMP for 2-16 h at room temperature. The mixturewas then purified by preparative HPLC and the fractions containing purepeptide combined and lyophilized. In the case of compounds with anotherlysine protected by an ivDde group, the deprotection reaction employed2% hydrazine in DMSO for 3 h at room temperature. Purification of thereaction mixture afforded pure peptide.

In the case of preparing a compound with S-acetylthioglycolic acidcoupled to two aminodioxaoctanoic acid groups and the peptide, thepurified peptide from Method 5 (having a free amino group, was coupledto AcSCH₂—CO—(NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO)₂—OH (30 eq.)/HOBt (30eq.)/DIC (30 eq.) in NMP for 40 h at room temperature. The mixture waspurified and the ivDde group was removed. A second purification gave thefinal product as a white lyophilizate.

Alternatively Fmoc aminodioxaoctanoic acid was coupled twicesuccessively to the peptide (produced by method 5) followed by Fmocremoval and coupling to S-acetylthioglycolic acid.

Method 13 Preparation of Homodimers and Heterodimers

The required purified peptides were prepared by SPPS using Method 5. Toprepare homodimers, half of the peptide needed to prepare the dimer wasdissolved in DMF and treated with 10 equivalents of glutaric acid bisN-hydroxysuccinimidyl ester The progress of the reaction was monitoredby HPLC analysis and mass spectroscopy. At completion of the reaction,the volatiles were removed in vacuo and the residue was washed withethyl acetate to remove the unreacted bis-NHS ester. The residue wasdried, re-dissolved in anhydrous DMF and treated with another halfportion of the peptide in the presence of 2 equivalents of DIEA. Thereaction was allowed to proceed for 24 hr. This mixture was applieddirectly to a Waters Associates C-18 XTerra RP-HPLC column and purifiedby elution with a linear gradient of acetonitrile into water (bothcontaining 0.1% TFA).

In the case of heterodimers, one of the monomers was reacted with thebis NHS ester of glutaric acid and after washing off the excess of bisNHS ester, the second amine was added in the presence of DIEA. After thereaction, the mixture was purified by preparative HPLC.

Preparation of KDR and VEGF/KDR Complex Binding Polypeptides

Utilizing the methods described above, the KDR and VEGF/KDR complexbinding polypeptides in Table 1 were prepared. As used in Table 1, theletter “J” in the peptide sequences refers to the spacer or linkergroup, 8-amino-3,6-dioxaoctanoyl. Also as used in Table 1, thedesignation “C*” refers to a cysteine residue that contributes to adisulfide bond. The ability of the biotinylated polypeptides to bind toKDR was assessed using the assay set described below.

The following biotinylated peptides bound well to the KDR-expressingcells: P13-XB (Kd 1.81 nM+/−0.27), P5-XB (Kd 14.87+/−5.07 nM, fourexperiment average), P6-XB (Kd 10.00+/−2.36 nM, four experimentaverage), P12-XB (Kd 4.031+/−0.86 nM, three experiment average), P6-F-XB(Kd 6.94+/−1.94 nM, one experiment), and P12-F-XB (Kd 3.02+/−0.75 nM,one experiment).

TABLE 1 Sequence or Structure of Peptides and Peptide Derivatives Ref.  SEQ.ID Number Structure or Sequence NO P1 Contol Peptide P1-BBiotinylated Control Peptide P1-XBBiotinylated Control Peptide with Spacer P2 AGWIECYHPDGICYHFGT 1 P2-DAc-AGWIEC*YHPDGIC*YHFGTGGGK-NH₂ P3 AGWLECYAEFGHCYNFGT 2 P3-DAc-AGWLEC*YAEFGHC*YNFGTGGGK-NH₂ P4 AGDSWCSTEYTYCEMIGT 3 P4-DAc-AGDSWC*STEYTYC*EMIGT-GGGK-NH₂ P5 AGPKWCEEDWYYCMITGT 4 P5-DAc-AGPKWC*EEDWYYC*MITGT-GGGK-NH₂ P5-EAc-AGPK(ivDde)WC*EEDWYYC*MITGTGGGK-NH₂ P5-BAc-AGPKWC*EEDWYYC*MITGT-GGGK-(Biotin)-NH₂ P5-XBAc-AGPKWC*EEDWYYC*MITGTGGGK-(Biotin-JJ-)-NH₂ P6 GDSRVCWEDSWGGEVCFRYDP 5P6-D Ac-GDSRVC*WEDSWGGEVC*FRYDPGGGK-NH₂ P6-BAc-GDSRVC*WEDSWGGEVC*FRYDP-GGGK-(Biotin)-NH₂ P6-XBAc-GDSRVC*WEDSWGGEVC*FRYDPGGGK-(Biotin-JJ-)-NH₂ P6-F-XBAc-VC*WEDSWGGEVC*FRYDPGGGK-(Biotin-JJ-)-NH₂ P7 GDWWECKREEYRNTTWCAWADP 6P7-D Ac-GDWWEC*KREEYRNTTWC*AWADPGGGK-NH₂ P7-EAc-GDWWEC*K(ivDde)REEYRNTTWC*AWADPGGGK-NH₂ P8 GDPDTCTMWGDSGRWYCFPADP 7P8-D Ac-GDPDTC*TMWGDSGRWYC*FPADPGGGK-NH₂ P9 AQEPEGYAYWEVITLYHEEDGDGG 8P9-D Ac-AQEPEGYAYWEVITLYHEEDGDGGK-NH₂ P10 AQAFPRFGGDDYWIQQYLRYTDGG 9P10-D Ac-AQAFPRFGGDDYWIQQYLRYTDGGK-NH₂ P11 AQGDYVYWEIIELTGATDHTPPGG 10P11-D Ac-AQGDYVYWEIIELTGATDHTPPGGGK-NH₂ P12 AGPTWCEDDWYYCWLFGT 11 P12-DAc-AGPTWC*EDDWYYC*WLFGT-NH₂ P12-XBAc-AGPTWC*EDDWYYC*WLFGT-GGGK-(Biotin-JJ-)-NH₂ P12-F-XBAc-AGPTWCEDDWYYCWLFGTJK-(Biotin-JJ-)-NH₂ P12-CAc-AGPTWC*EDDWYYC *WLFGTGGGKJJGC(Acm)- (N,N-dimethyl-G SC (Acm)-NH₂ P13AQDWYYDEILSMADQLRHAFLSGG 12 P13-D Ac-AQDWYYDEILSMADQLRHAFLSGG-NH₂ P13-XBAc-AQDWYYDEILSMADQLRHAFLS GG-GGGK-(Biotin-JJ-)-NH₂ P14GSDHHCYLHNGQWICYPFAPGGGK 13 P14-D Ac-GSDHHC*YLHNGQWIC*YPFAPGGGK-NH₂ P15GDYPWCHELSDSVTRFCVPWDPGGGK 14 P15-D Ac-GDYPWC*HELSDSVTRFC*VPWDPGGGK-NH₂P16 GDDHMCRSPDYQDHVFCMYWDPGGGK 15 P16-DAc-GDDHMC*RSPDYQDHVFC*MYWDPGGGK-NH₂ P17 GDPPLCYFVGTQEWHHCNPFDPGGGK 16P17-D Ac-GDPPLC*YFVGTQEWHHC*NPFDPGGGK-NH₂ P18 GDGSWCEMRQDVGKWNCFSDDPGGGK17 P18-E Ac-GDGSWC*EMRQDVGK(-ivDde-)WNC*FSDDPGGGK-NH₂ P19AQRGDYQEQYWHQQLVEQLKLLGGGK 18 P19-EAc-AQRGDYQEQYWHQQLVEQLK(-ivDde-)LLGGGK-NH₂ P20GDNWECGWSNMFQKEFCARPDPGGGK 19 P20-EAc-GDNWEC*GWSNMFQK(-ivDde-)EFC*ARPDPGGGK-NH₂ P21AGPGPCK(-ivDde-)GYMPHQCWYMGTGGGK  20 P21-EAc-AGPGPC*K(-ivDde-)GYMPHQC*WYMGTGGGK-NH₂ P22 AGYGPCAEMSPWLCWYPGTGGGK 21P22-D Ac-AGYGPC*AEMSPWLC*WYPGTGGGK-NH₂

Example 2 Bead-Binding Assay to Confirm Ability of Peptides Identifiedby Phage Display to Bind KDR-Expressing Cells

The following experiments were performed to assess the ability ofKDR-binding peptides to bind to KDR-expressing cells. In thisexperiment, KDR-binding peptides P5-B and P5-XB and P6-B and P6-XB wereconjugated to fluorescent beads and their ability to bind toKDR-expressing 293H cells was assessed. The experiments show that bothpeptide sequences can be used to bind particles such as beads toKDR-expressing sites. In general, the P6 peptides exhibited betterbinding to the KDR expressing cells than P5. However, the binding ofboth peptides improved with the addition of a spacer.

Biotinylation of an Anti-KDR Antibody

Anti-KDR from Sigma (V-9134), as ascites fluid, was biotinylated using akit from Molecular Probes (F-6347) according to the manufacturer'sinstructions.

Preparation of Peptide-Conjugated Fluorescent Beads

0.1 mL of a 0.2 mM stock solution of each biotinylated peptide (preparedas set forth above, in 50% DMSO) was incubated with 0.1 mL ofNeutravidin-coated red fluorescent microspheres (2 micron diameter,custom-ordered from Molecular Probes) and 0.2 mL of 50 mM MES (SigmaM-8250) buffer, pH 6.0 for 1 hour at room temperature on a rotator. As apositive control, biotinylated anti-KDR antibody was incubated with theNeutravidin-coated beads as above, except that 0.03 mg of thebiotinylated antibody preparation in PBS (Gibco 14190-136) was usedinstead of peptide solution. Beads can be stored at 4° C. until neededfor up to 1 week.

Transfection of 293H Cells

293H cells were transfected using the protocol described in Example 6.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. # 354640). The cells in one half of the plate (48 wells) weremock-transfected (with no DNA) and those in the other half of the platewere transfected with KDR cDNA. The cells were 80-90% confluent at thetime of transfection and completely confluent the next day, at the timeof the assay; otherwise the assay was aborted.

Binding Assay

From the above bead preparations, 0.12 mL was spun for 10 minutes at2000 rpm in a microcentrifuge at room temperature. The supernatant wasremoved and 0.06 mL of MES pH 6.0 was added. Each bead solution was thenvortexed and sonicated in a water bath 15 min. To 1.47 mL of DMEM, highglucose (GIBCO 11965-084) with 1×MEM Non-Essential Amino Acids Solution(NEAA) (GIBCO 11140-050) and 40% FBS (Hyclone SH30070.02) 0.03 mL of thesonicated bead preparations was added. 96-well plates seeded with 293Hcells which have been mock-transfected in columns 1 to 6, andKDR-transfected in columns 7 to 12 (as described above), were drainedand washed once with DMEM, high glucose with 1×NEAA and 40% FBS. To eachwell was added 0.1 mL of bead solution, six wells per bead preparation.After incubating at room temperature for 30 minutes, the wells weredrained by inverting the plates and washed four times with 0.1 mL PBSwith Ca⁺⁺Mg⁺⁺ (GIBCO 14040-117) with shaking at room temperature for 5minutes each wash. After draining, 0.1 mL of PBS was added per well. Theplates were then read on a Packard FluoroCount fluorometer at excitation550 nm/emission 620 nm. Unconjugated Neutravidin beads were used as anegative control while beads conjugated with a biotinylated anti-KDRantibody were used as the positive control for the assay.

To calculate the number of beads bound per well, a standard curve withincreasing numbers of the same fluorescent beads was included in eachassay plate. The standard curve was used to calculate the number ofbeads bound per well based on the fluorescence intensity of each well.

As shown in FIG. 1, the positive control beads with anti-KDR attachedclearly bound preferentially to the KDR-expressing cells while avidinbeads with nothing attached did not bind to either cell type.Biotinylated P5 beads did not bind to the KDR-transfected cellssignificantly more than to mock-transfected cells, but adding ahydrophilic spacer between the peptide moiety and the biotin groupenhanced binding to KDR cells without increasing the binding tomock-transfected cells. Biotinylated P6 beads showed greater binding toKDR-transfected cells. As was the case for P5, adding a hydrophilicspacer between the peptide portion and the biotin of the moleculesignificantly improved the specific binding to KDR in the transfectedcells. Thus the peptide sequences of both P5 and P6 can be used to bindparticles such as beads to KDR expressing sites.

Example 3 Competition of KDR Binding Peptides and ¹²⁵I-Labeled VEGF forBinding to KDR-Transfected 293H Cells

The following experiment assesses the ability of KDR-binding peptides tocompete with ¹²⁵I-labeled VEGF for binding to KDR expressed bytransfected 293H cells. While KDR-binding polypeptide P4 did not competesignificantly with ¹²⁵I-labeled VEGF, P5-XB, P6 and P12-XB competed verywell with ¹²⁵I-labeled VEGF, inhibiting 96.29±2.97% and 104.48±2.07% of¹²⁵I-labeled VEGF binding.

Transfection of 293H Cells

293H cells were transfected using the protocol described in Example 6.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. # 354640). The cells in one half of the plate (48 wells) weremock-transfected (with no DNA) and those in the other half of the platewere transfected with KDR cDNA. The cells were 80-90% confluent at thetime of transfection and completely confluent the next day, at the timeof the assay; otherwise the assay was aborted.

Preparation of M199 Media

To prepare M199 medium for the assay, one M199 medium packet (GIBCO,cat. # 31100-035), 20 mL of 1 mM HEPES (GIBCO, cat. #15630-080), and 2 gof DIFCO Gelatin (DIFCO, cat. # 0143-15-1) were added to 950 mL ofdouble distilled (dd) H₂O and the pH of the solution was adjusted to 7.4by adding approximately 4 mL of 1N NaOH. After pH adjustment, the M199medium was warmed to 37° C. in a water bath for 2 h to dissolve thegelatin, then filter sterilized using 0.2 μm filters (Corning, cat. #43109), and stored at 4° C. to be used later in the assay.

Preparation of Peptide Solutions

3 mM stock solutions of peptides P6, P4, P5-XB, and P12-XB, (prepared asdescribed above) in 50% DMSO were prepared.

Preparation of ¹²⁵I-Labeled VEGF Solution for the Assay

25 μCi of lyophilized ¹²⁵I-labeled VEGF (Amersham, cat. # IM274) werereconstituted with 250 μL, of ddH₂O to create a stock solution, whichwas stored at −80° C. for later use. For each assay, a 300 μM solutionof ¹²⁵I-labeled VEGF was made fresh by diluting the above stock solutionin M199 medium. The concentration of ¹²⁵I-labeled VEGF was calculateddaily based on the specific activity of the material on that day.

Preparation of 30 μM and 0.3 μM Peptide Solution in 300 μM ¹²⁵I-LabeledVEGF

For each 96 well plate, 10 mL of 300 μM ¹²⁵I-labeled VEGF in M199 mediumwas prepared at 4° C. Each peptide solution (3 mM, prepared as describedabove) was diluted 1:100 and 1:10000 in 300 μL, of M199 media with 300μM ¹²⁵I-labeled VEGF to prepare 30 μM and 0.3 μM peptide solutionscontaining 300 μM of ¹²⁵I-labeled VEGF. Once prepared, the solutionswere kept on ice until ready to use. The dilution of peptides in M199media containing 300 μM ¹²⁵I-labeled VEGF was done freshly for eachexperiment.

Assay to Detect Competition with ¹²⁵I-Labeled VEGF in 293H Cells

Cells were used 24 h after transfection, and to prepare the cells forthe assay, they were washed 3 times with room temperature M199 mediumand placed in the refrigerator. After 15 minutes, the M199 medium wasremoved from the plate and replaced with 75 μL, of 300 μM ¹²⁵I-labeledVEGF in M199 medium (prepared as above). Each dilution was added tothree separate wells of mock and KDR transfected cells. After incubatingat 4° C. for 2 h, the plates were washed 5 times with cold bindingbuffer, gently blotted dry and checked under a microscope for cell loss.100 μL, of solubilizing solution (2% Triton X-100, 10% Glycerol, 0.1%BSA) was added to each well and the plates were incubated at roomtemperature for 30 minutes. The solubilizing solution in each well wasmixed by pipeting up and down, and transferred to 1.2 mL tubes. Eachwell was washed twice with 100 μL, of solubilizing solution and thewashes were added to the corresponding 1.2 mL tube. Each 1.2 mL tube wasthen transferred to a 15.7 mm×10 cm tube to be counted in an LKB GammaCounter (¹²⁵I window for 1 minute).

Competition of Peptides with ¹²⁵I-Labeled VEGF in 293H Cells

The ability of KDR-binding peptides P6, P4, P5-XB, and P12-XB, tospecifically block ¹²⁵I-labeled VEGF binding to KDR was assessed inmock-transfected and KDR-transfected cells. P4 was used in the assay asa negative control. It was selected because it exhibits only poorbinding to KDR in FP assays, and thus would not be expected to displaceor compete with VEGF. To calculate the specific binding to KDR, thebinding of ¹²⁵I-labeled VEGF to mock-transfected cells was subtractedfrom KDR-transfected cells. Therefore, the binding of ¹²⁵I-labeled VEGFto sites other than KDR (which may or may not be present in 293H cells)is not included when calculating the inhibition of ¹²⁵I-labeled VEGFbinding to 293H cells by KDR-binding peptides.

FIG. 2 shows the percentage inhibition of ¹²⁵I-labeled VEGF binding bypeptides (P6, P4, P5-XB, and P12-XB) at two different concentrations (30μM and 0.3 μM) to KDR-transfected 293H cells. Percentage inhibition wascalculated using formula [(Y1-Y2)×100/Y1], where Y1 is specific bindingto KDR-transfected 293H cells in the absence of peptides, and Y2 isspecific binding to KDR-transfected 293H cells in the presence ofpeptides or DMSO (vehicle). Specific binding to KDR-transfected 293Hcells was calculated by subtracting binding to mock-transfected 293Hcells from binding to KDR-transfected 293H cells. Results for P6, P4 andP5-XB are the average of three experiments±SD, whereas the result forP12-XB is from one experiment.

As shown in FIG. 2, P4, which, due to its relatively high Kd (>2 μM,measured by FP against KDR-Fc), was used as a negative control, did notcompete significantly with ¹²⁵I-labeled VEGF, 12.69+7.18% at 30 μM and−5.45±9.37% at 0.3 μM (FIG. 2). At the same time, P6, and P12-XBcompeted very well with ¹²⁵I-labeled VEGF, inhibiting 96.29±2.97% and104.48±2.07% of ¹²⁵I-labeled VEGF binding at 30 μM and 52.27±3.78% and80.96±3.8% at 0.3 μM, respectively. The percentage inhibition withP5-X-B was 47.95±5.09% of ¹²⁵I-labeled VEGF binding at 30 μM and24.41±8.43% at 0.3 μM (FIG. 2). Thus, as one would expect, a peptidethat only binds KDR poorly did not block VEGF binding, while three otherKDR-binding peptides did compete with VEGF, and their potency increasedwith their binding affinity. This assay should also be useful foridentifying peptides that bind tightly to KDR but do not compete withVEGF, a feature that may be useful for imaging KDR in tumors, wherethere is frequently a high local concentration of VEGF that wouldotherwise block the binding of KDR-targeting molecules.

Example 4 Inhibition of VEGF-Induced KDR Receptor Activation by PeptidesIdentified by Phage Display

The ability of KDR-binding peptides identified by phage display toinhibit VEGF induced activation (phosphorylation) of KDR was assessedusing the following assay. A number of peptides of the invention wereshown to inhibit activation of KDR in monomeric and/or tetramericconstructs, including P5-D, P6-D, P10-D and P11-D. As discussed above,peptides that inhibit activation of KDR may be useful as anti-angiogenicagents.

Human umbilical vein endothelial cells (HUVECs) (Biowhittaker Cat No.CC-2519) were obtained frozen on dry ice and stored in liquid nitrogenuntil thawing. These cells were thawed, passaged, and maintained asdescribed by the manufacturer in EGM-MV medium (Biowhittaker Cat No.CC-3125). Cells seeded into 100 mm dishes were allowed to becomeconfluent, then cultured overnight in basal EBM medium lacking serum(Biowhittaker Cat No. CC-3121). The next morning, the medium in thedishes was replaced with 10 mL fresh EBM medium at 37° C. containingeither no additive (negative control), 5 ng/mL VEGF (Calbiochem Cat No.676472 or Peprotech Cat No. 100-20) (positive control), or 5 ng/mL VEGFplus the indicated concentration of the KDR-binding peptide (prepared asdescribed above). In some cases, a neutralizing anti-KDR antibody (CatNo. AF357, R&D Systems) was used as a positive control inhibitor ofactivation. In such cases, the antibody was pre-incubated with the testcells for 30 min at 37° C. prior to the addition of fresh mediumcontaining both VEGF and the antibody. After incubating the dishes 5 minin a 37° C. tissue culture incubator they were washed three times withice-cold Dulbecco's phosphate buffered saline (D-PBS) containing calciumand magnesium and placed on ice without removing the last 10 mL ofD-PBS. The first dish of a set was drained and 0.5 mL of Triton lysisbuffer was added (20 mM Tris base pH 8.0, 137 mM NaCl, 10% glycerol, 1%Triton X-100, 2 mM EDTA (ethylenediaminetetraacetic acid), 1 mM PMSF(phenylmethylsulfonylfluoride), 1 mM sodium orthovanadate, 100 mM NaF,50 mM sodium pyrophosphate, 10 μg/mL leupeptin, 10 μg/mL aprotinin). Thecells were quickly scraped into the lysis buffer using a cell scraper(Falcon, Cat No. 353087), dispersed by pipeting up and down briefly, andthe resulting lysate was transferred to the second drained dish of thepair. Another 0.5 mL of lysis buffer was used to rinse out the firstdish then transferred to the second dish, which was then also scrapedand dispersed. The pooled lysate from the two dishes was transferred toa 1.5 mL Eppindorf tube. The above procedure was repeated for each ofthe controls and test samples (KDR-binding peptides), one at a time. Thelysates were stored on ice until all the samples had been processed. Atthis point samples were either stored at −70° C. or processed to the endof the assay without interruption.

The lysates, either freshly prepared or frozen and thawed, wereprecleared by adding 20 μL of protein A-sepharose beads (Sigma 3391,preswollen in D-PBS), washed three times with a large excess of D-PBS,reconstituted with 6 mL D-PBS to generate a 50% slurry) and rocked at 4°C. for 30 min. The beads were pelleted by centrifugation for 2 min in aPicofuge (Stratgene, Cat No. 400550) at 2000xg and the supernatantstransferred to new 1.5 mL tubes. 20 μg of anti-Flk-1 antibody (SantaCruz Biotechnology, Cat No. sc-504) was added to each tube, and thetubes were incubated overnight (16-18 h) at 4° C. on a rotator toimmunoprecipitate KDR. The next day 40 μL of protein A-sepharose beadswere added to the tubes, which were then incubated at 4° C. for 1 h on arotator. The beads in each tube were subsequently washed three times bycentrifuging for 2 min in a Picofuge, discarding the supernatant, anddispersing the beads in 1 mL freshly added TBST buffer (20 mM Tris basepH 7.5, 137 mM NaCl, and 0.1% Tween 20). After centrifuging and removingthe liquid from the last wash, 40 μL of Laemmli SDS-PAGE sample buffer(Bio-Rad, Cat No. 161-0737) was added to each tube and the tubes werecapped and boiled for 5 min. After cooling, the beads in each tube werepelleted by centrifuging and the supernatants containing theimmunoprecipitated KDR were transferred to new tubes and usedimmediately or frozen and stored at −70° C. for later analysis.

Detection of phosphorylated KDR as well as total KDR in theimmunoprecipitates was carried out by immunoblot analysis. Half (20 μL)of each immunoprecipitate was resolved on a 7.5% precast Ready Gel(Bio-Rad, Cat No. 161-1154) by SDS-PAGE according to the method ofLaemmli (U. K. Laemmli “Cleavage of structural proteins during assemblyof the head of bacteriophage T4.” Nature (1970); 227, 680-685).

Using a Bio-Rad mini-Protean 3 apparatus (Cat No. 165-3302). Theresolved proteins in each gel were electroblotted to a PVDF membrane(Bio-Rad, Cat. No. 162-0174) in a Bio-Rad mini Trans-Blot cell (Cat No.170-3930) in CAPS buffer (10 mM CAPS, Sigma Cat No. C-6070, 1% ACS grademethanol, pH 11.0) for 2 h at 140 mA according to the method ofMatsudaira (P. Matsudaira. “Sequence from picomole quantities ofproteins electroblotted onto polyvinylidine diflouride membranes.” J.Biol. Chem. (1987); 262, 10035-10038). Blots were blocked at roomtemperature in 5% Blotto-TBS (Pierce Cat No. 37530) pre-warmed to 37° C.for 2 h. The blots were first probed with an anti-phosphotyrosineantibody (Transduction Labs, Cat No. P11120), diluted 1:200 in 5%Blotto-TBS with 0.1% Tween 20 added for 2 h at room temp. The unboundantibody was removed by washing the blots four times with D-PBScontaining 0.1% Tween 20 (D-PBST), 5 min per wash. Subsequently, blotswere probed with an HRP-conjugated sheep anti-mouse antibody (AmershamBiosciences Cat No. NA931) diluted 1:25,000 in 5% Blotto-TBS with 0.1%Tween 20 added for 1 h at room temperature, and washed four times withD-PBST. Finally, the blots were incubated with 2 mL of achemiluminescent substrate (ECL Plus, Amersham Cat No. RPN2132) spreadon top for 2 min, drip-drained well, placed in plastic sheet protector(C-Line Products, Cat No. 62038), and exposed to X-ray film (KodakBioMax ML, Cat No. 1139435) for varying lengths of time to achieveoptimal contrast.

To confirm that similar amounts of KDR were compared in the assay, theblots were stripped by incubating for 30 min at 37° C. in TBST with itspH adjusted to 2.4 with HCl, blocked for 1 h at room temp with 5%Blotto-TBS with 0.1% Tween 20 (Blotto-TBST), and reprobed with ananti-Flk-1 polyclonal antibody (Cat No. sc-315 from Santa Cruz Biotech),1:200 in 5% Blotto-TBST with 1% normal goat serum (Life Tech Cat No.16210064) for 2 h at room temp. The unbound antibody was removed bywashing the blots four times with D-PBST, 5 min per wash. Subsequently,the blots were probed with an HRP-conjugated donkey anti-rabbitsecondary antibody (Amersham Biosciences Cat No. NA934) diluted 1:10,000in 5% Blotto-TBST for 1 h at room temperature, and washed four timeswith D-PBST. Finally, the blots were incubated with 2 mL ofchemiluminescent substrate and exposed to X-ray film as described above.

Immunoblots of KDR immunoprecipitates prepared from HUVECs with andwithout prior VEGF stimulation, shown in FIG. 3, demonstrated thatactivated (phosphorylated) KDR could be detected when the HUVECs werestimulated with VEGF. An anti-phosphotyrosine antibody (PY-20) detectedno phosphorylated proteins close to the migration position of KDR fromunstimulated HUVECs on the blots, but after five minutes of VEGFstimulation, an intense band was consistently observed at the expectedlocation (FIG. 3, upper panel). When the blots were stripped of boundantibodies by incubation in acidic solution then reprobed with ananti-KDR antibody (sc-315), the identity of the phosphorylated proteinband was confirmed to be KDR. Moreover, it was observed thatimmunopreciptates from unstimulated HUVECs contained about as much totalKDR as immunoprecipitates from VEGF-stimulation HUVECs (FIG. 3, lowerpanel).

It is reasonable to conclude that the phosphorylated KDR detected wasformed from pre-existing KDR through autophosphorylation of KDR dimersresulting from VEGF binding as five minutes is not enough time tosynthesize and process a large glycosylated cell-surface receptor suchas KDR.

The ability of the assay to detect agents capable of blocking the VEGFactivation of KDR was assessed by adding a series of compounds to HUVECsin combination with VEGF and measuring KDR phosphorylation with theimmunoblot assay described above. As negative and positive controls,immunoprecipitates from unstimulated HUVECs and from HUVECs stimulatedwith VEGF in the absence of any test compounds were also tested in everyassay. When a neutralizing anti-KDR antibody (Cat No. AF-357 from R&DSystems) was combined with the VEGF, the extent of KDR phosphorylationwas greatly reduced (FIG. 4, upper panel), indicating that the antibodywas able to interfere with the ability of VEGF to bind to and activateKDR. This result was expected because the ability of the antibody toblock VEGF-induced DNA synthesis is part of the manufacturer's qualitycontrol testing of the antibody. Re-probing the blot with an anti-KDRantibody (FIG. 4, lower panel) indicated that slightly less total KDRwas present in the VEGF+antibody-treated lane (+V+α-KDR) relative to theVEGF-only-treated lane (+V), but the difference was not great enough toaccount for the much lower abundance of phosphorylated KDR in theantibody-treated lane.

To assess the potency of a KDR-binding peptide (P10-D) identified byphage display, the experiment was repeated with P10-D in the presence ofVEGF. P10-D was able to largely inhibit the VEGF-induced phosphorylationof KDR. Re-probing the blot for total KDR showed that there is even moretotal KDR in the VEGF+P10-D-treated cells (+V+P10-D) than in the VEGFonly-treated cells (+V) (FIG. 5, lower panel). Thus, it is clear thatthe decreased phosphorylation of KDR in the presence of P10-D is not dueto differential sample loading, but rather the ability of the compoundto inhibit VEGF-activation of KDR.

Using the methods of this Example, the following peptides demonstratedat least a 50% inhibition of VEGF-induced KDR phosphorylation at 10 μM:

P2-D, P3-D, P6-D, P7-E, P8-D, P9-D, P10-D, P11-D.

P2 and P6 were the most potent compounds in the assay, producing atleast a 50% inhibition of VEGF-induced KDR phosphorylation at 1 μM.

The following peptides were tested in the assay and did not producesignificant inhibition of KDR activation at 10 μM:

P5-E, P14-D, P15-D, P16-D, P17-D, P18-E, P19-E, P20-E, P21-E, P23-D

In addition, tetrameric complexes of biotinylated derivatives P6-XB orP12-XB (prepared as described above and discussed in Example 6, infra)produced at least a 50% inhibition of VEGF-induced KDR phosphorylationat 10 nM.

Example 5 Binding of Tc-Labeled Polypeptide to KDR-Transfected 293HCells

In this Example, the ability of Tc-labeled P12-C to bind KDR wasassessed using KDR-transfected 293H cells. The results show thatTc-labeled P12-C bound significantly better to KDR transfected 293Hcells than to mock transfected 293H cells, and binding increased withconcentration of the Tc-labeled polypeptide in a linear manner.

Preparation of Peptidic Chelate (P12-C) for Binding to Tc by SPPS

To a 250 ml of SPPS reaction vessel was added 6.64 mmol ofH-Gly-2-Cl-trityl resin (0.84 mmol/g, Novabiochem). It was swelled in 80mL of DMF for 1 h. For each coupling cycle the resin was added 26.6 mmolof DIEA, 26.6 mmol of a Fmoc-amino acid in DMF (EM Science), 26.6 mmolof HOBT (Novabiochem) in DMF, and 26.6 mmol of DIC. The total volume ofDMF was 80 mL. The reaction mixture was shaken for 4 h. The resin thenwas filtered and washed with DMF (3×80 mL). A solution of 20% piperidinein DMF (80 mL) was added to the resin and it was shaken for 10 min. Theresin was filtered and this piperidine treatment was repeated. The resinfinally was washed with DMF (3×80 mL) and ready for next coupling cycle.At the last coupling cycle, N,N-dimethyl glycine (Aldrich) was coupledusing HATU/DIEA activation. Thus, to a suspension of N,N-dimethylglycine (26.6 mmol) in DMF was added a solution of 26.6 mmol of HATU(Perseptive Biosystems) in DMF and 53.1 mmol of DIEA. The clear solutionwas added to the resin and shaken for 16 h. Following the synthesis, theresin was filtered and washed with DMF (3×80 mL), CH₂Cl₂ (3×80 ml) anddried. The resin was mixed with 80 mL of AcOH/CF₃CH₂OH/DCM (1/1/8,v/v/v) and shaken for 45 min. The resin was filtered and the filtratewas evaporated to a paste. Purification of the crude material by silicagel chromatography using 25% MeOH/DCM afforded 2.0 g of the finalproduct.

Coupling of the Peptidic Chelate (P12-C) to the Peptide (FragmentCoupling)

To a mixture of purified Me₂N-Gly-Cys-(Trt)-Ser(tBu)-Gly-OH— andhydroxybenzotriazole (0.0055 mmol) in DMF (0.25 mL),diisopropylcarbodiimide (0.0055 mmol) was added and the mixture wasstirred at RT for 6 h. The peptide (0.005 mmol) in DMF (0.25 mL) wasthen added to the reaction mixture and stirring was continued for anadditional 6 h. DMF was removed under vacuum and the residue was treatedwith reagent B and stirred for 3 h. TFA was removed under reducedpressure and the residue was purified by preparative HPLC usingacetonitrile-water containing 0.1% TFA. Fractions containing the pureproduct were collected and freeze dried to yield the peptide. Thepeptide was characterized by ES-MS and the purity was determined byRP-HPLC (acetonitrile-water/0.1% TFA) gradient.

Synthesis of ^(99m)Tc-Labeled Peptide

A stannous gluconate solution was prepared by adding 2 mL of a 20 μg/mLSnCl₂.2H₂O solution in nitrogen-purged 1N HCl to 1.0 mL ofnitrogen-purged water containing 13 mg of sodium glucoheptonate. To a 4mL autosampler vial was added 20-40 μL (20-40 μg) of P12-C liganddissolved in 50/50 ethanol/H₂O, 6-12 mCi of ^(99m)TcO₄ ⁻ in saline and100 μL of stannous glucoheptonate solution. The mixture was heated at100° C. for 22 min. The resulting radiochemical purity (RCP) was 10-47%when analyzed using a Vydac C18 Peptide and Protein column that waseluted at a flow rate of 1 mL/min with 66% H₂O (0.1% TFA)/34% ACN(0.085%TFA). The reaction mixture was purified by HPLC on a Vydac C18 column(4.6 mm×250 mm) at a flow rate of 1 mL/min, using 0.1% TFA in water asaqueous phase and 0.085% TFA in acetonitrile as the organic phase. Thefollowing gradient was used; 29.5% org. for 35 min., ramp to 85% org.over 5 min, hold for 10 min. The fraction containing ^(99m)Tc-P12-C(which no longer contained the ACM protecting group) was collected into500 μL of a stabilizing buffer containing 5 mg/mL ascorbic acid and 16mg/mL hydroxypropyl-γ-cyclodextrin in 50 mM phosphate buffer. Themixture was concentrated using a speed vacuum apparatus to removeacetonitrile, and 200 μL of 0.1% HSA in 50 mM pH 5 citrate buffer wasadded. The resulting product had an RCP of 100%. Prior to injection intoanimals, the compound was diluted to the desired radioconcentration withnormal saline.

Transfection of 293H Cells

293H cells were transfected using the protocol described in avidin HRPexample. Transfection was done in black/clear 96-well plates (BectonDickinson, cat. # 354640). The cells in one half of the plates (48wells) were mock-transfected (with no DNA) and the cells in the otherhalf of the plate were transfected with KDR cDNA. The cells were 80-90%confluent at the time of transfection and completely confluent the nextday, at the time of the assay; otherwise the assay was aborted.

Preparation of opti-MEMI Media with 0.1% HSA

Opti-MEMI was obtained from Invitrogen (cat. # 11058-021) and humanserum albumin (HSA) was obtained from Sigma (cat. # A-3782). To prepareopti-MEMI media with 0.1% HSA, 0.1% w/v HSA was added to opti-MEMI,stirred at room temperature for 20 minutes, and then filter sterilizedusing 0.2 μM filter.

Preparation of Tc-Labeled Peptide Dilutions for the Assay

Stock solution of Tc-labeled P12-C (117 μCi/ml) was diluted 1:100, 1:50,1:25 and 1:10 in opti-MEMI with 0.1% HSA to provide solutions with finalconcentration of 1.17, 2.34, 4.68 and 11.7 μCi/mL of Tc-labeled P12-C

Assay to Detect the Binding of Tc-Labeled Peptide

Cells were used 24 h after transfection, and to prepare the cells forthe assay, they were washed 1× with 100 μL of room temperature opti-MEMIwith 0.1% HSA. After washing, the opti-MEMI with 0.1% HSA was removedfrom the plate and replaced with 70 μL of 1.17, 2.34, 4.68 and 11.7μCi/mL of Tc-labeled P12-C (prepared as above). Each dilution was addedto three separate wells of mock and KDR transfected cells. Afterincubating at room temperature for 1 h, the plates were transferred to4° C. for 15 minutes and washed 5 times with 100 μL of cold bindingbuffer (opti-MEMI with 0.1% HSA), gently blotted dry and checked under amicroscope for cell loss. 100 μL of solubilizing solution (2% TritonX-100, 10% Glycerol, 0.1% BSA) was added to each well and the plateswere incubated at 37° C. for 10 minutes. The solubilizing solution ineach well was mixed by pipeting up and down, and transferred to 1.2 mLtubes. Each well was washed once with 100 μL of solubilizing solutionand the washes were added to the corresponding 1.2 mL tube. Each 1.2 mLtube was then transferred to a 15.7 mm×100 cm tube to be counted in anLKB Gamma Counter (Tc-window for 20 sec).

Binding of Tc-Labeled Peptide to KDR Transfected Cells

The ability of Tc-labeled P12-C to bind specifically to KDR wasdemonstrated using transiently transfected 293H cells. As shown in FIG.6, Tc-labeled P12-C bound significantly better to KDR transfected 293Hcells, as compared to mock transfected 293H cells. To calculate specificbinding to KDR, the binding of Tc-labeled P12-C to mock transfectedcells was subtracted from the binding to KDR transfected cells. As shownin FIG. 7, a linear increase in the specific binding of Tc-labeled P12-Cto KDR was observed with increasing concentration of Tc-labeled P12-C.Linear binding was expected because the concentration of Tc-labeledP12-C was only ˜100 pM (even at the highest concentration, 11.7 μCi/mL,tested in the assay), which is far below the K_(D) value of ˜3-4 nM ofP12 (as calculated using the avidin HRP assay), so no saturation ofbinding would be expected.

Example 6 Binding of KDR Binding Peptide/Avidin HRP Complex to KDRTransfected 293H Cells

To determine the binding of peptides identified by phage display to KDRexpressed in transiently-transfected 293H cells, a novel assay thatmeasures the binding of biotinylated peptides complexed with neutravidinHRP to KDR on the surface of the transfected cells was developed. Thisassay was used to screen the biotinylated peptides described above.Neutravidin HRP was used instead of streptavidin or avidin because ithas lower non-specific binding to molecules other than biotin, due tothe absence of lectin binding carbohydrate moieties and also due to theabsence of the cell adhesion receptor-binding RYD domain in neutravidin.

In the experiments described herein, tetrameric complexes of KDR-bindingpeptides P6-XB, P5-XB, P12-XB, and P13-XB and a control peptide, P1-XB,were prepared and tested for their ability to bind 293H cells that weretransiently-transfected with KDR. All four tetrameric complexes ofKDR-binding peptides bound to the KDR-expressing cells; however, P13-XBexhibited the best Kd (1.81 nM). The tetrameric complexes of KDR-bindingpeptides P6-XB and P5-XB exhibited improved binding over monomers of thesame peptides. Moreover, inclusion of a spacer (between the KDR-bindingpeptide and the biotin) in these constructs was shown to improve bindingin Experiment B.

Experiment C, demonstrates the use of this assay to assess the effect ofserum on binding of peptides of the invention to KDR and VEGF/KDRcomplex. The binding of P5-XB, P6-XB and P13-XB was not significantlyaffected by the presence of serum, while the binding of P12-XB wasreduced more than 50% in the presence of serum.

Experiment D demonstrates that this assay is useful in evaluatingdistinct combinations of KDR and VEGF/KDR complex binding polypeptidesfor use in multimeric targeting constructs which contain more than oneKDR and VEGF/KDR complex binding polypeptide. Moreover, Experiments Dand E establish that heteromeric constructs, which have two or more KDRbinding peptides that bind to different binding sites, exhibitedsuperior binding to “homotetrameric” constructs of the targetingpeptides alone.

Experiment A Preparation of m-RNA and 5′ RACE Ready cDNA Library

HUVEC cells were grown to almost 80% confluence in 175 cm² tissueculture flasks (Becton Dickinson, Biocoat, cat #6478) and then 10 ng/mLof bFGF (Oncogene, cat #PF003) was added for 24 h to induce expressionof KDR. MRNA was isolated using the micro-fast track 2.0 kit fromInvitrogen (cat. # K1520-02). 12 μg of mRNA (measured by absorbance at260 nM) was obtained from two flasks (about 30 million cells) followingthe kit instructions. Reverse transcription to generate cDNA wasperformed with 2 μg of mRNA, oligo dT primer (5′-(T)₂₅GC-3′) and/orsmart II oligo (5′AAGCAGTGGTAACAACGCAGAGTA CGCGGG-3′) using MoloneyMurine Leukemia Virus (MMLV) reverse transcriptase. The reaction wasperformed in a total volume of 20 μL and the reaction mix contained 2 μLof RNA, 1 μL smart II oligo, 1 μL of oligo dT primer, 4 μL of 5×first-strand buffer (250 mM Tris HCl pH 8.3, 375 mM KCl, 30 mM MgCl₂) 1μL DTT (20 mM, also supplied with reverse transcriptase), 1 μL dNTP mix(10 mM each of dATP, dCTP, dGTP, and dTTP in ddH₂O, Stratagene, cat. #200415), 9 μL ddH₂O and 1 μL MMLV reverse transcriptase (Clonetech, cat#8460-1). The reverse transcription reaction was performed for 90minutes at 42° C., and the reaction was stopped by adding 250 μL oftricine-EDTA buffer (10 mM tricine, 1.0 mM EDTA). The reversetranscription product, a 5′ RACE ready cDNA library, can be stored for 3months at −20° C. All water used for DNA and RNA applications was DNAseand RNAse free from USB (cat. # 70783).

Cloning of s-KDR into TOPOII Vector

To clone s-KDR, a 5′ oligo (G ATG GAG AGC AAG GTG CTG CTG G) and a 3′oligo (C CAA GTT CGT CTT TTC CTG GGC A) were used. These were designedto amplify the complete extracellular domain of KDR (˜2.2 kbps) from the5′ RACE ready cDNA library (prepared above) using polymerase chainreaction (PCR) with pfu polymerase (Stratagene, cat. # 600135). The PCRreaction was done in total volume of 50 μL and the reaction mixcontained 2 μL 5′ RACE ready cDNA library, 1 μL 5′ oligo (10 μM), 1 μL3′ oligo (10 μM), 5 μL 10×PCR buffer [PCR buffer (200 mM Tris-HCl pH8.8, 20 mM MgSO₄, 100 mM KCl, 100 mM (NH₄)₂SO₄) supplied with pfu enzymeplus 1% DMSO and 8% glycerol], 1 μL dNTP mix (10 mM) and 40 μL ddH₂O.The PCR reaction was performed by using a program set for 40 cycles of 1minute at 94° C., 1 minute at 68° C. and 4 minutes at 72° C. The PCRproduct was purified by extraction with 1 volume of phenol, followed byextraction with 1 volume of chloroform and precipitated using 3 volumeof ethanol and 1/10 volume of 3M sodium acetate. The PCR product wasresuspended in 17 μL of ddH₂O, the 2 μL of 10×Taq polymerase buffer (100mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl₂, 0.01% gelatin) and 1 μL ofTaq polymerase (Stratagene, cat. # 600131) was added to generate an Aoverhang to each end of the product. After incubating for 1 hour at 72°C. the modified product was cloned directly into a TOPOII vector frominvitrogen (cat. # K4600-01) following the manufacturer's protocol togive TOPO-sKDR. The TOPO vector allows easy cloning of PCR productsbecause of the A-overhang in Taq (PCR enzyme)-treated PCR products.

Cloning the Transmembrane and Cytoplasmic Domains of KDR into TOPO IIVector

To clone the transmembrane and cytoplasmic domains of KDR, a 5′ oligo(TCC CCC GGG ATC ATT ATT CTA GTA GGC ACG GCG GTG) and a 3′ oligo (C AGGAGG AGA GCT CAG TGT GGT C) were used. These were designed to amplify thecomplete transmembrane and cytoplasmic domains of KDR (˜1.8 kbps) fromthe 5′ RACE ready cDNA library (described above) using polymerase chainreaction (PCR) with pfu polymerase. PCR reaction conditions and theprogram were exactly the same as described above for s-KDR. Just as withthe s-KDR sequence, the PCR product was purified using phenol chloroformextraction, treated with Taq polymerase and cloned into TOPOII vectorfrom invitrogen to give TOPO-CYTO.

Cloning of Full-Length KDR into pcDNA6 Vector

To create the full-length receptor, the extra-cellular domain and thecytoplasmic domain (with trans-membrane domain) were amplified by PCRseparately from TOPO-sKDR and TOPO-CYTO respectively and ligated laterto create the full-length receptor. An oligo with a Not1 site at the 5′end of the extracellular domain (A TAA GAA TGC GGC CGC AGG ATG GAG AGCAAG GTG CTG CTG G) and an oligo complimentary to the 3′ end of theextracellular domain (TTC CAA GTT CGT CTT TTC CTG GGC ACC) were used toamplify by PCR the extracellular domain from TOPO-sKDR. Similarly, the5′ oligo (ATC ATT ATT CTA GTA GGC ACG GCG GTG) and the 3′ oligo, with aNot1 site (A TAA GAA TGC GGC CGC AAC AGG AGG AGA GCT CAG TGT GGT C),were used to amplify by PCR the cytoplasmic domain of KDR (withtransmembrane domain) from TOPO-CYTO. Both PCR products were digestedwith Not1 and ligated together to create the full-length receptor. ThecDNA encoding the full-length receptor was purified on an agarose geland ligated into the Not1 site of the pcDNA6/V5-His C vector.Purification of DNA and ligation was done as described earlier forpsKDR. The ligation reaction was used to transform a culture of DH5αbacteria and a number of individual clones were analyzed for thepresence and orientation of insert by restriction analysis of purifiedplasmid from each clone with EcoRI enzyme.

Cell Culture

293H cells were obtained from Invitrogen (cat. # 11631) and grown asmonolayer cultures in their recommended media plus 1 mL/L pen/strep(Invitrogen, cat. # 15140-148). All the cells were grown in presence ofantibiotic for everyday culture but were split into antibiotic freemedia for 16-20 hour prior to transfection.

Preparation of DNA for Transfection

E. coli. bacteria DH5α containing pf-KDR was streaked onto LB with 50μg/mL ampicillin (LB agar from US biologicals, cat. # 75851 andampicillin from Sigma, cat. #A2804) plates from a glycerol stock andplates were left in a 37° C. incubator to grow overnight. Next morning,a single colony was picked from the plate and grown in 3 mL ofLB/ampicillin media (LB from US biologicals, cat. # US75852) at 37° C.After 8 hours, 100 μL of bacterial culture from the 3 mL tube wastransferred to 250 mL of LB/ampicillin media for overnight incubation at37° C. Bacteria were grown up with circular agitation in a 500 mL bottle(Beckman, cat. # 355605) at 220 rpm in a Lab-Line incubator shaker. Thenext day, the bacterial culture was processed using maxi-prep kit(QIAGEN, cat. # 12163). Generally, about 1 mg of plasmid DNA (asquantitated by absorbance at 260 nm) was obtained from 250 mL ofbacterial culture.

Transfection of 293H Cells in 96 Well Plate

Transfection was done as recommended in the lipofectamine 2000 protocol(Invitrogen, cat# 11668-019) using a poly-D-lysine-coated 96 well plate.320 ng of KDR DNA (pc-DNA6-fKDR)/per well in 0.1 mL was used for 96 wellplate transfections. Transfection was done in serum-containing media,the transfection reagent mix was removed from cells after 6-8 hours andreplaced with regular serum-containing medium. Transfection was done inblack/clear 96-well plates (Becton Dickinson, cat. # 354640). The cellin one half of the plate (48 wells) were mock-transfected (with no DNA)and the cells in the other half of the plate were transfected with KDRcDNA. The cells were 80-90% confluent at the time of transfection andcompletely confluent next day, at the time of the assay, otherwise theassay was aborted.

Preparation of M199 Media

M199 media was prepared as described above

Preparation of Softlink Soft Release Avidin-Sepharose

SoftLink soft release avidin-sepharose was prepared by centrifuging thesepharose obtained from Promega (cat. # V2011) at 12,000 rpm for 2minutes, washing twice with ice cold water (centrifuging in-between thewashes) and resuspending the pellet in ice cold water to make a 50%slurry in ddH₂O. A fresh 50% slurry of avidin-sepharose was prepared foreach experiment.

Preparation of Peptide/Neutravidin Hrp Solution

Biotinylated peptides P6-XB, P5-XB, P12-XB, P13-XB and the biotinylatedcontrol peptide, P1-XB, (prepared as described above) were used toprepare 250 μM stock solutions in 50% DMSO and a 33 μM stock solution ofNeutravidin HRP was prepared by dissolving 2 mg of Neutravidin HRP(Pierce, cat. # 31001) in 1 mL of ddH₂O. Peptide stock solutions werestored at −20° C., whereas the Neutravidin HRP stock solution was storedat −80° C. The structures of the biotinylated peptides are shown inTable 1. To prepare peptide/neutravidin HRP complexes, 10 μL of 250 μMbiotinylated peptide stock solution and 10 μL of 33 μM Neutravidin HRPwere added to 1 ml of M199 medium. This mixture was incubated on arotator at 4° C. for 60 minutes, followed by addition of 50 μL of softrelease avidin-sepharose (50% slurry in ddH₂O) to remove excess peptidesand another incubation for 30 minutes on a rotator at 4° C. Finally, thesoft release avidin-sepharose was pelleted by centrifuging at 12,000 rpmfor 5 minutes at room temperature, and the resulting supernatant wasused for the assays. Fresh peptide/neutravidin HRP complexes wereprepared for each experiment.

Preparation of Peptide/Neutravidin Hrp Dilutions for the Assay

For saturation binding experiments, 120 μL, 60 μL, 20 μL, 10 μL, 8 μL, 6μL, 4 μL and 1 μL of peptide/neutravidin HRP complex were added to 1.2ml aliquots of M199 medium to create dilutions with final concentrationsof 33.33 nM, 16.65 nM, 5.55 nM, 2.78 nM, 1.67 nM, 1.11 nM and 0.28 nMcomplex, respectively.

Preparation of Blocking Solution for Transfected 293H Cells

Blocking solution was prepared by adding 20 mL of M199 medium to 10 mgof lyophilized unlabeled neutravidin (Pierce, cat. # 31000). Freshblocking solution was used for each experiment.

Assay to Detect the Binding of Peptide/Neutravidin HRP

24 h after transfection, each well of the 293H cells was washed 1× with100 μL of M199 medium and incubated with 80 μL of blocking solution at37° C. After one hour, cells were washed 2× with 100 μL of M199 mediaand incubated with 70 μL of peptide/neutravidin HRP dilutions of P1-XB,P6-XB, P5-XB, P12-XB and P13-XB for two and half hours at roomtemperature. Each dilution was added to three separate wells of mock aswell as KDR-transfected 293H cells (two plates were used for eachsaturation binding experiment). After incubation at room temperature,plates were transferred to 4° C. for another half-hour incubation.Subsequently, cells were washed five times with ice-cold M199 media and1× with ice-cold PBS (in that order). After the final wash, 100 μL ofice cold TMB solution (KPL, cat. # 50-76-00) was added to each well andeach plate was incubated for 30 minutes at 37° C. in an air incubator.Finally, the HRP enzyme reaction was stopped by adding 50 μL of 1Nphosphoric acid to each well, and binding was quantitated by measuringabsorbance at 450 nm using a microplate reader (BioRad Model 3550).

Binding of Peptide/Neutravidin HRP to KDR-Transfected Cells

In this assay, complexes of P6-XB, P5-XB, P12-XB, P13-XB peptides, andthe control peptide, P1-XB, with neutravidin HRP were prepared asdescribed above and tested for their ability to bind 293H cells thatwere transiently-transfected with KDR. During the peptide/neutravidincomplex preparation, a 7.5 fold excess of biotinylated peptides overneutravidin HRP was used to ensure that all four biotin binding sites onneutravidin were occupied. After complex formation, the excess of freebiotinylated peptides was removed using soft release avidin-sepharose toavoid any competition between free biotinylated peptides and neutravidinHRP-complexed biotinylated peptides.

The experiment was performed at several different concentrations ofpeptide/neutravidin HRP, from 0.28 nM to 33.33 nM, to generatesaturation binding curves for P5-XB and P6-XB (FIG. 8A) and 0.28 to 5.55nM to generate saturation binding curve for P12-XB and P13-XB (FIG. 8B).To draw the saturation binding curve, the background binding tomock-transfected cells was subtracted from the binding toKDR-transfected cells for each distinct peptide/neutravidin HRP complexat each concentration tested. Therefore, absorbance on the Y-axis ofFIG. 8 is differential absorbance (KDR minus mock) and not the absoluteabsorbance. Analysis of the saturation binding data in FIG. 8 usingGraph Pad Prism software (version 3.0) yielded a Kd of 10.00 nM(+/−2.36) for the tetrameric P6-XB, 14.87 nM (+/−5.07) for thetetrameric P5-XB, 4.03 nM (+/−0.86) for the tetrameric P12-XB and 1.81nM (+/−0.27) for the tetrameric P13-XB peptide complexes. These bindingconstants are, as expected, lower than those measured by FP against theKDRFc construct for the related monodentate peptides P6 (69 nM) and P5(280 nM) (fluoresceinated) but similar for the monodentate peptide P12(3 nM). As expected, no saturation of binding for the control P1-X-Bpeptide/neutravidin HRP-complex was observed. As shown in FIG. 9, thebinding of peptide/neutravidin HRP complexes at a single concentration(5.55 nM) was plotted to demonstrate that a single concentrationexperiment can be used to differentiate between a KDR binding peptide(P6-XB, P5-XB and P12-XB) from a non-binding peptide (P1-XB).

Experiment B

Experiment B was designed to look at the effect of a spacer (X) betweenthe KDR binding sequence (P6 and P5) and biotin. In this experiment,biotinylated P6 and P5 (with and without spacer X, prepared as set forthabove) were tested, and P1 (with and without spacer, prepared as setforth above) was used as a negative control.

This experiment was performed as set forth in Experiment A describedabove, except that it was only done at single concentration of 2.78 nM.It is evident from the results, shown in FIG. 10, that a spacer (X) isrequired for effective binding of P6 and P5. The spacer (X) between thebinding sequence and biotin can be helpful in enhancing binding totarget molecule by multiple mechanisms. First, it may help reduce thesteric hindrance between four biotinylated peptides after their bindingto a single avidin molecule. Second, it may provide extra lengthnecessary to reach multiple binding sites available on a single cell.

Experiment C

Experiment C examined the serum effect on the binding of P6-XB, P5-XB,P12-XB, and P13-XB. In this experiment, biotinylated peptide/avidin HRPcomplexes of P6-XB, P5-XB, P12-XB, and P13-XB were tested in M199 media(as described above in Experiment A) with and without 40% rat serum.This experiment was performed as described for Experiment A, except thatit was only done at single concentration of 6.66 nM for P6-XB and P5-XB,3.33 nM for P12-XB and 2.22 nM for P13XB.

The results, shown in FIG. 11, indicate that binding of P6-XB, P5-XB andP13-XB was not significantly affected by 40% rat serum whereas bindingof P12-XB dropped more than 50% in presence of 40% rat serum. More thanan 80% drop in the binding of Tc-labeled P12-C(P12 with Tc-chelate),prepared by the method described in Example 5 above, was observed in thepresence of 40% rat serum (data shown in FIG. 25). Because the serumeffect on the binding of Tc-labeled P12-C is mimicked in the avidin HRPassay disclosed herein, this assay may be used to rapidly evaluate theserum effect on the binding of peptide(s) to KDR.

Experiment D

Experiment D was designed to evaluate the binding of tetramericcomplexes of KDR and VEGF/KDR complex-binding polypeptides P6-XB andP5-XB, particularly where the constructs included at least two KDRbinding polypeptides. The KDR binding peptides and control bindingpeptide (P1-XB) were prepared as described above. This experiment wasperformed using the protocol set forth for Experiment A, except theprocedures set forth below were unique to this experiment.

Preparation of Peptide/Neutravidin Hrp Solutions

250 μM stock solutions of biotinylated peptides P1-X-B, P6-XB, and P5-XBwere prepared in 50% DMSO and a 33 μM stock solution of Neutravidin HRPwas prepared by dissolving 2 mg of Neutravidin HRP (Pierce, cat. #31001) in 1 mL of ddH₂O. Peptide stock solutions were stored at −20° C.,whereas the Neutravidin HRP stock solution was stored at −80° C. Toprepare peptide/neutravidin HRP complexes, a total 5.36 μL of 250 μMbiotinylated peptide stock solution (or a mixture of peptide solutions,to give peptide molecules four times the number of avidin HRP molecules)and 10 μL of 33 μM Neutravidin HRP were added to 1 mL of M199 medium.This mixture was incubated on a rotator at 4° C. for 60 minutes,followed by addition of 50 μL of soft release avidin-sepharose (50%slurry in ddH₂0) to remove excess peptides and another incubation for 30minutes on a rotator at 4° C. Finally, the soft release avidin-sepharosewas pelleted by centrifuging at 12,000 rpm for 5 minutes at roomtemperature, and the resulting supernatant was used for the assays.Fresh peptide/neutravidin HRP complexes were prepared for eachexperiment.

Assay to Detect the Binding of Peptide/Neutravidin HRP

The procedure described above was used to detect binding of thepeptide/neutravidin HRP. The results of this experiment establish thatP6-XB and P5-XB bind to KDR in multimeric fashion, and cooperate witheach other for binding to KDR in 293H transfected cells.

P1-XB is a biotinylated derivative of P1, a control peptide that doesnot bind to KDR. As expected, a tetrameric complex of P1-XB withavidin-HRP did not show enhanced binding to KDR-transfected cells. Asshown in FIG. 12, tetrameric complexes of P6-XB or P5-XB bound toKDR-transfected cells significantly better than to mock-transfectedcells. P6-XB tetramers however, bound much better than P5-X tetramers.When P1-XB was added to the peptide mixture used to form the tetramericcomplexes, the binding to the KDR-transfected cells was decreased. Theratios of specific binding of tetramer to monomer, dimer and trimer werecalculated by dividing the specific binding (obtained by subtracting thebinding to mock transfected cells from KDR transfected cells) oftetramer, trimer & dimer with that of monomer. Results suggest thatthere is co-operative effect of multimerization of P5-XB, P6-XB andP13-XB on the binding to KDR transfected cells.

TABLE 2 Enhanced binding of homomultimeric constructs over monomers Ref.Number Tetramer Trimer Dimer P5-XB 45.4 5 4.3 P6-XB* 38.6 7.1 2.7 P12-XB1 1.1 1.1 P13-XB 16 5.7 2.3 *Monomeric Peptide binding at 3.33 nM waszero and therefore the ratios were calculated using binding at 5.55 nM.

A mixture of 25% P1-XB with 75% P5-XB did not bind significantly overbackground to KDR-transfected cells, indicating that multivalent bindingis critical for the P5-XB/avidn-HRP complex to remain bound to KDRthroughout the assay. This phenomenon also held true for P6-XB, wheresubstituting 50% of the peptide with P1-XB in the tetrameric complexabolished almost all binding to KDR on the transfected cells.

A peptide mixture composed of 50% P1-XB with 25% P6-XB and 25% P5-XBbound quite well to KDR-transfected cells relative to mock-transfectedcells, indicating that there is a great advantage to targeting twobinding sites on the same target molecule. Furthermore, it was notedthat tetrameric complexes containing different ratios of P6-XB and P5-XB(3:1, 2:2, and 1:3) all bound much better to KDR-transfected cells thanpure tetramers of either peptide, in agreement with the idea thattargeting two distinct sites on a single target molecule is superior tomultimeric binding to a single site. This may be because multimericbinding to a single target requires that the multimeric binding entityspan two or more separate target molecules which are close enoughtogether for it to bind them simultaneously, whereas a multimeric binderwhich can bind two or more distinct sites on a single target moleculedoes not depend on finding another target molecule within its reach toachieve multimeric binding. The ratios of specific binding ofheterotetramer, heterotrimer and heterodimer to monomer were calculatedby dividing the specific binding (obtained by subtracting the binding tomock transfected cells from KDR transfected cells) of tetramer, trimerand dimer with that of monomer. Monomer, which was used to calculate theratios, for each set of heteromers is recorded at the end of eachheteromer listing in the table and given the ratio of 1.

TABLE 3 Enhanced binding of heteromultimeric constructs over monomersPeptide Mix Heteromer/ Where (1X, 2X, 3X) is the occupancy Monomer Conc.of the four avidin sites Ratio (nM) P6-XB (1X) + P5-XB (3X) 529 3.33P6-XB (2X) + P5-XB (2X) 777 3.33 P6-XB (3X) + P5-XB (1X) 633 3.33 P1-XB(1X) + P6-XB (1X) + P5-XB (2X) 213 3.33 P1-XB (1X) + P6-XB (2X) + P5-XB(1X) 242 3.33 P1-XB (2X) + P6-XB (1X) + P5-XB (1X) 109 3.33 P5-XB (1X) +P1-XB (3X) 1 3.33 P6-XB (1X) + P12-XB (3X) 46 2.78 P6-XB (2X) + P12-XB(2X) 42 2.78 P6-XB (3X) + P12-XB (1X) 43 2.78 P1-XB (1X) + P6-XB (1X) +P12-XB (2X) 47 2.78 P1-XB (1X) + P6-XB (2X) + P12-XB (1X) 52 2.78 P1-XB(2X) + P6-XB (1X) + P12-XB (1X) 40 2.78 P1-XB (3X) + P6-XB (1X)* 1 5.55P1-XB (1X) + P13-XB (1X) + P12-XB (2X) 5 2.78 P1-XB (1X) + P13-XB (2X) +P12-XB (1X) 7 2.78 P1-XB (2X) + P13-XB (1X) + P12-XB (1X) 2 2.78 P13-XB(1X) + P1-XB (3X) 1 2.78 P1-XB (1X) + P6-XB (1X) + P13-XB (2X) 83 2.78P1-XB (1X) + P6-XB (2X) + P13-XB (1X) 31 2.78 P1-XB (2X) + P6-XB (1X) +P13-XB (1X) 31 2.78 P1-XB (3X) + P6-XB (1X)* 1 5.55

The enhanced binding ratios of the homodimers range from about 1-4 foldas seen in table 2 whereas the binding of the heterodimers ranges from2-110 fold, demonstrating the synergistic effect on binding strength ofcomplementary sequences (Table 3).

Experiment E

Experiment E was designed to confirm that P6-XB and P5-XB bind todistinct binding sites on KDR. If the peptides bind to the same site onKDR, they would likely compete with each other for binding to KDR,whereas if the peptides bind to different sites, there should be nocompetition between the peptides for binding to KDR. This experiment wasperformed using a single concentration of P5-XB/avidin HRP (3.33 nM)solution in each well and adding a varying concentration (0-2.5 μM) ofP1-XB, P5-XB and P6-XB, none of which were complexed with avidin.

It is evident from the results, shown in FIG. 13, that P5-XB doescompete with P5-XB/avidin HRP solution for binding to KDR transfectedcells whereas P1-XB and P6-XB do not compete with P5-XB/avidin HRPsolution for binding to KDR transfected cells. Thus, P5-XB and P6-XBbind to distinct and complementary sites on KDR.

Example 7 Preparation of Heterodimeric Constructs

To obtain a higher affinity peptide binder to the KDR receptor, twolinear peptides (P9, P10) were linked together to form a heterodimer. Asdetermined by VEGF competition assays, these two peptides bind differentsites on KDR. It is possible, therefore, that both peptides in theheterodimer could bind a single protein molecule at the same time and asresult, bind with a higher overall affinity for the receptor. Two formsof the heterodimer were synthesized in an effort to determine theoptimal orientation for this bidentate binding event. The peptides wereeither linked together in a tail-to-tail orientation via theirC-terminal lysine residues or in a head-to-head orientation via theirN-terminal amino groups.

The peptides were synthesized using standard Fmoc solid-phase peptidesynthesis protocols. To add spacing between the two peptides in thedimer, each individual peptide monomer was modified at either theC-terminal lysine (to make the tail-to-tail dimer) or N-terminal amino(to make the head-to-head dimer) with a monodispersed PEG-based aminoacid linker (Fmoc-NH-PEG₄-CO₂H). After deprotection of the Fmoc group ofeach PEG linker, the P9 peptide was labeled with levulinic acid(CH₃(C═O)(CH₂)₂CO₂H) and the P10 peptide was labeled withBoc-amino-oxyacetic acid. After deprotection, cleavage and purification,the two peptides were ligated together in a 1:1 ratio in denaturingbuffer (8M Urea, 0.1M sodium acetate, pH 4.6) to form an oxime linkage(—CH═N—O—) between the two different peptides. Using the two differentsets of monomers, the tail-to-tail and head-to-head heterodimers wereformed in solution and purified to homogeneity by standard reverse phaseprotocols. A more detailed description of this linkage chemistry isfound in K. Rose, et al. JACS, 1999, 121: 7034-7038, which is herebyincorporated by reference in its entirety.

Assay for Binding Affinity of Heterodimeric Constructs

To assay for improved binding affinity relative to either monomericpeptide, each heterodimer was assayed for binding using a surfaceplasmon resonance instrument (Biacore 3000). Soluble KDR receptor wascross-linked to the dextran surface of a CM5 sensor chip by the standardamine coupling procedure. A 0.5 mg/mL solution was diluted 1:40 with 50mM acetate, pH 6.0 to immobilize a total R_(L), of 12721. Experimentswere performed in PBST buffer (5.5 mM phosphate, pH 7.65, 0.15M NaCl,0.1% Tween-20 (v/v)). Peptide solutions quantified by extinctioncoefficient were diluted to produce 1000, 500, 250, 125, 62.5 and 31.3nM solutions. For association, peptides were injected at 20 μL/min for 2minutes using the kinject program. Following a 3 minute dissociation,any remaining peptide was stripped from the KDR surface with aquickinject of 50 mM NaOH, 1M NaCl for 15 s at 75 μL/min. Monomeric P9and P10 were run as standards. Sensorgrams were analyzed by globalanalysis using BIAevaluation software 3.1.

The peptide dimers investigated in this study by BIAcore analysis bindKDR with significantly higher affinity than either of the constituentmonomers. By design, the interaction of a dimeric peptide with KDR isexpected to proceed through two kinetic steps. With more detailedanalysis it may be possible to accurately dissect the individual rateconstants for these steps. However, an apparent K_(D) was calculated forthe dimer interaction using the rate describing the initial encounter(k_(a,1)) and the predominant off-rate (k_(d,2)). From this analysis,the apparent K_(D) of the head-to-head dimer was 2.2 nM and that of thetail-to-tail dimer was 11 nM (Table 4). These estimates represent anincrease in affinity over the individual monomers of greater than60-fold for the comparison of the P9 to T-T dimer K_(D) (732 nM/11.1 nM)and greater than 560-fold for the P10 to H—H dimer K_(D) (1260 nM/2.24nM).

TABLE 4 Summary of Kinetic Parameters Peptide k_(a, 1) (1/Ms) k_(d, 1)(1/s) K_(D, 1) (nM) Chi2* P9 4.53 × 10³ 3.32 × 10⁻³ 732 0.67 P10 3.60 ×10⁵  4.5 × 10⁻¹ 1260 1.2 Head-to-head dimer 1.11 × 10⁵ 2.49 × 10⁻⁴ 2.241.25 Tail-to-tail dimer 1.15 × 10⁵ 1.28 × 10⁻³ 11.1 2.33

Example 8

The following methods were used for the preparation of individualpeptides and dimeric peptide constructs described in Examples (8-12).

Automated Peptide Synthesis

Peptide synthesis was carried out on an ABI-433A Synthesizer (AppliedBiosystems Inc., Foster City, Calif.) on a 0.25 mmol scale using theFastMoc protocol. In each cycle of this protocol preactivation wasaccomplished by dissolution of 1.0 mmol of the requisite dry N^(α)-Fmocside-chain protected amino acid in a cartridge with a solution of 0.9mmol of HBTU, 2 mmol of DIEA, and 0.9 mmol of HOBt in a DMF-NMP mixture.The peptides were assembled on NovaSyn TGR (Rink amide) resin(substitution level 0.2 mmol/g). Coupling was conducted for 21 min. Fmocdeprotection was carried out with 20% piperidine in NMP. At the end ofthe last cycle, the N-terminal Fmoc group was removed and the fullyprotected resin-bound peptide was acetylated using aceticanhydride/DIEA/HOBt/NMP.

Cleavage, Side-chain Deprotection and Isolation of Crude Peptides

Cleavage of the peptides from the resin and side-chain deprotection wasaccomplished using Reagent B for 4.5 h at ambient temperature. Thecleavage solutions were collected and the resins were washed with anadditional aliquot of Reagant B. The combined solutions wereconcentrated to dryness. Diethyl ether was added to the residue withswirling or stirring to precipitate the peptides. The liquid phase wasdecanted, and solid was collected. This procedure was repeated 2-3 timesto remove impurities and residual cleavage cocktail components.

Cyclization of Di-Cysteine Peptides

The crude ether-precipitated linear di-cysteine containing peptides werecyclized by dissolution in water, mixtures of aqueous acetonitrile (0.1%TFA), aqueous DMSO or 100% DMSO and adjustment of the pH of the solutionto 7.5-8.5 by addition of aqueous ammonia, aqueous ammonium carbonate,aqueous ammonium bicarbonate solution or DIEA. The mixture was stirredin air for 16-48 h, acidified to pH 2 with aqueous trifluoroacetic acidand then purified by preparative reverse phase HPLC employing a gradientof acetonitrile into water. Fractions containing the desired materialwere pooled and the purified peptides were isolated by lyophilization.

Preparation of Peptides Containing Linkers

In a typical experiment, 400 mg of the resin-bound peptide bearing anivDde-protected lysine) was treated with 10% hydrazine in DMF (2×20 mL).The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). The resin wasresuspended in DMF (10 mL) and treated withFmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC(0.4 mmol) and DIEA (0.8 mmol) with mixing for 4 h. After the reaction,the resin was washed with DMF (2×10 mL) and with DCM (1×10 mL). Theresin was then treated with 20% piperidine in DMF (2×15 mL) for 10 mineach time. The resin was washed and the coupling withFmoc-8-amino-3,6-dioxaoctanoic acid and Fmoc protecting group removalwere repeated once more.

The resulting resin-bound peptide with a free amino group was washed anddried and then treated with reagent B (20 mL) for 4 h. The mixture wasfiltered and the filtrate concentrated to dryness. The residue wasstirred with ether to produce a solid, which was washed with ether anddried. The solid was dissolved in anhydrous DMSO and the pH adjusted to7.5 with DIEA. The mixture was stirred for 16 h to effect the disulfidecyclization and the reaction was monitored by analytical HPLC. Aftercompletion of the cyclization, the reaction mixture was diluted with 25%acetonitrile in water and applied directly to a reverse phase C-18column. Purification was effected using a gradient of acetonitrile intowater (both containing 0.1% TFA). Fractions were analyzed by HPLC andthose containing the pure product were combined and lyophilized toprovide the required peptide.

Preparation of Biotinylated Peptides Containing Linkers

In a typical experiment, 400 mg of the resin-bound peptide bearing anivDde-protected lysine, was treated with 10% hydrazine in DMF (2×20 mL).The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). The resin wasresuspended in DMF (10 mL) and treated withFmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC(0.4 mmol) and DIEA (0.8 mmol) with mixing for 4 h. After the reaction,the resin was washed with DMF (2×10 mL) and with DCM (1×10 mL). Theresin was then treated with 20% piperidine in DMF (2×15 mL) for 10 mineach time. The resin was washed and the coupling withFmoc-8-amino-3,6-dioxaoctanoic acid and removal of the Fmoc protectinggroup were repeated once more.

The resulting resin-bound peptide with a free amino group was treatedwith a solution of Biotin-NHS ester (0.4 mmol, 5 equiv.) and DIEA (0.4mmol, 5 equiv.) in DMF for 2 h. The resin was washed and dried asdescribed previously and then treated with Reagent B (20 mL) for 4 h.The mixture was filtered and the filtrate concentrated to dryness. Theresidue was stirred with ether to produce a solid that was collected,washed with ether, and dried. The solid was dissolved in anhydrous DMSOand the pH adjusted to 7.5 with DIEA. The mixture was stirred for 4-6 hto effect the disulfide cyclization which was monitored by HPLC. Uponcompletion of the cyclization, the reaction mixture was diluted with 25%acetonitrile in water and applied directly to a reverse phase C-18column. Purification was effected using a gradient of acetonitrile intowater (both containing 0.1% TFA). Fractions were analyzed by HPLC andthose containing the pure product were collected and lyophilized toprovide the required biotinylated peptide.

Preparation of DOTA-Conjugated Peptides for Labeling with SelectedGadolinium or Indium Isotopes

In a typical experiment, 400 mg of the resin-bound peptide bearing anN^(ε)-ivDde-protected lysine moiety was treated with 10% hydrazine inDMF (2×20 mL). The resin was washed with DMF (2×20 mL) and DCM (1×20mL). The resin was resuspended in DMF (10 mL) and treated withFmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC(0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. After the reaction, theresin was washed with DMF (2×10 mL) and with DCM (1×10 mL). The resinwas then treated with 20% piperidine in DMF (2×15 mL) for 10 min eachtime. The resin was washed and the coupling withFmoc-8-amino-3,6-dioxaoctanoic acid and removal of the Fmoc protectinggroup were repeated once. The resulting resin-bound peptide with a freeamino group was resuspended in DMF (10 mL) and treated with a solutionof 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid,-1,4,7-tris-t-butyl ester (DOTA-tris-t-butyl ester, 0.4 mmol, 5equiv.), HOBt (0.4 mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) in DMF (10mL) with mixing for 4 h. Upon completion of the reaction, the resin waswashed with DMF (2×10 mL) and with DCM (1×10 mL) and treated withReagent B (20 mL) for 4 h. The mixture was filtered and the filtrateconcentrated to dryness. The residue was stirred in ether to produce asolid that was collected, washed with ether, and dried. The solid wasdissolved in anhydrous DMSO and the pH adjusted to 7.5 with DIEA. Themixture was stirred for 16 h to effect the disulfide cyclization, whichwas monitored by HPLC. Upon completion of the cyclization, the mixturewas diluted with 25% acetonitrile in water and applied directly to areverse phase C-18 HPLC column. Purification was effected using agradient of acetonitrile into water (both containing 0.1% TFA).Fractions were analyzed by HPLC and those containing the pure productwere combined and lyophilized to provide the required biotinylatedpeptide.

The following monomeric peptides of Table 5 were prepared by the abovemethods.

TABLE 5Sequence or Structure of Monomeric Peptides and Peptide Derivatives SeqRef. Id. Number Structure or Sequence No: P12-XB-KAc-AGPTWC*EDDWYYC*WLFGTGGGK(BiotinJJ-K)-NH₂ — P12-XDT-KAc-AGPTWC*EDDWYYC*WLFGTJK(DOTAJJ-K)-NH₂ — P12-XAc-AGPTWC*EDDWYYC*WLFGTJK(JJ)-NH₂ — P12-EAc-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH₂ — P6-F-XB-KAc-VC*WEDSWGGEVC*FRYDPGGGK(Biotin-JJ-K)-NH₂ — P6-F-XAc-VC*WEDSWGGEVC*FRYDPGGGK(JJ)-NH₂ — P13-EB-KAc-AQDWYYDEILSMADQLRHAFLSGGGGGK(ivDde)K(Biotin-JJ)-NH₂ — P13-XAc-AQDWYYDEILSMADQLRHAFLSGGGGGK(J)-NH2 — P13-K-EAc-AQDWYYDEILSMADQLRHAFLSGGGGGKK(ivDde) — P6-XAc-GDSRVC*WEDSWGGEVC*FRYDPGGGK(JJ)-NH₂ — P12-AAc-AGPTWC*EDDWYYC*WLFGTGGGK[(PnAO6- — C(═O)(CH₂)₃C(═O)-K]-NH₂P12-XDT-K-E Ac-AGPTWC*EDDWYYC*WLFGTGGGK[DOTA-JJK(iV-Dde)]-NH₂ — P6-FAc-VC*WEDSWGGEVC*FRYDPGGGK-NH₂ — P12-OAc-AGPTWC*EDDWYYC*WLFGTGGGK[K(BOA)]-NH₂ — P13-A-EAc-AQDWYYDEILSMADQLRHAFLSGGGGGK[PnAO6- — C(═O)(CH₂)₃C(═O)-K(iV-Dde)]-NH₂P23 AQDWYYEILJGRGGRGGRGGK 22 P23-K-EAc-AQDWYYEILJGRGGRGGRGGK[K(ivDde)]-NH₂ P24 APGTWCDYDWEYCWLGTFGGGK 23P24-A Ac-APGTWC*DYDWEYC*WLGTFGGGK+(6PnAO- C(═O)(CH₂)₃C(═O)-K+-NH₂ P25GVDFRCEWSDWGEVGCRSPDYGGGK 24 P25-XAc-GVDFRC*EWSDWGEVGC*RSPDYGGGK(JJ)-NH₂ P12-BKAc-AGPTWC*EDDWYYC*WLFGTGGGK(Biotin-K)-NH₂ — P12-JEJJAGPTWC*EDDWYYC*WLFGTGGGK(iV-Dde)-NH₂ — P6-J-FJJVC*WEDSWGGEVC*FRYDPGGG-NH₂ — P12-JA[-JJAGPTWCEDDWYYCWLFGTGGGGK(PnAO6-Glut)-NH_(2])- — P12-SAc-AGPTWC*EDDWYYC*WLFGTGGGK[K(SATA)]-NH₂ — P12-SX-KAc-AGPTWC*EDDWYYC*WLFGTGGGK[SATA-JJ-K]-NH₂ — P12-NEH₂N-AGPTWC*EDDWYYC*WLFGTGGGK[K(iV-Dde)]-NH₂ — P12-QAc-AGPTWC*EDDWYYC*WLFGTGGGK{Biotin-JJK[NH₂- —Ser(GalNAc-alpha-D)-Gly-Ser(GalNAc₃-alpha-D]}-NH₂ — P6-F-QAc-VC*WEDSWGGEVC*FRYDPGGGK(NH₂-Ser(GalNAc-alpha- —D)-Gly-Ser(GalNAc-alpha-D)-NH₂ P26 GSPEMCMMFPFLYPCNHHAPGGGK 25 P26-AAc-GSPEMC*MMFPFLYPC*NHHAPGGGK[(P_(n)AO6)- C(═O)(CH2)3C(═O)-K]}-NH₂ P27GSFFPCWRIDRFGYCHANAPGGGK 26 P27-X Ac-GSFFPC*WRIDRFGYC*HANAPGGGK(JJ)-NH₂P27-A Ac-GSFFPC*WRIDRFGYC*HANAPGGGK[P_(n)AO6)- C(═O)(CH2)3C(═O)-K]}-NH₂P28 AQEWEREYFVDGFWGSWFGIPHGGGK 27 P28-XAc-AQEWEREYFVDGFWGSWFGIPHGGGK(JJ)-NH₂ P29 GDYSECFFEPDSFEVKCYDRDPGGGK 28P29-X Ac-GDYSEC*FFEPDSFEVKC*YDRDPGGGK(JJ)-NH₂

As used in Table 5 above and elsewhere herein, the designation “C*”refers to a cysteine residue that contributes to a disulfide bond. Ingeneral, the monomeric peptides described herein are prepared as cyclicdisulfide peptides and then linked together to form dimers.Consequently, even if a cysteine residue lacks the “C*” designation, thepresence of a disulfide bond to the nearest cysteine in the monomer cangenerally be assumed. The monomer components of the dimers will alsogenerally contain such disulfide bonds, regardless of whether thecysteine residues contain the “C*” designation or not. However, oneskilled in the art will appreciate that the dimers and otherheteromultimers of the present invention could alternatively be preparedby performing the cyclization of Di-cysteine peptides after the monomersare linked to form dimers, and the present invention is not intended tobe limiting with respect to the presence or absence of such disulfidebonds.

Example 9

The purified peptide monomers mentioned above in Example 8 were used inthe preparation of various homodimeric and heterodimeric constructs.

Preparation of Homodimer-Containing Constructs

To prepare homodimeric compounds, half of the peptide needed to preparethe dimer was dissolved in DMF and treated with 10 equivalents ofglutaric acid bis-N-hydroxysuccinimidyl ester. The progress of thereaction was monitored by HPLC analysis and mass spectroscopy. Atcompletion of the reaction, the volatiles were removed in vacuo and theresidue was washed with ethyl acetate to remove the unreacted bis-NHSester. The residue was dried, re-dissolved in anhydrous DMF and treatedwith another half portion of the peptide in the presence of 2equivalents of DIEA. The reaction was allowed to proceed for 24 h. Thismixture was applied directly to a YMC reverse phase HPLC column andpurified by elution with a linear gradient of acetonitrile into water(both containing 0.1% TFA).

Preparation of Heterodimer-Containing Constructs

In the case of heterodimers, one of the monomers (“A”) was reacted withthe bis-NHS ester of glutaric acid and after washing off the excess ofbis-NHS ester (as described for the homodimeric compounds), the secondmonomer (“B”) was added in the presence of DIEA. After the reaction themixture was purified by preparative HPLC. Typically, to a solution ofglutaric acid bis N-hydroxysuccinimidyl ester (0.02 mmol, 10equivalents) in DMF (0.3 mL) was added a solution of peptide A and DIEA(2 equiv) in DMF (0.5 mL) and the mixture was stirred for 2 h. Theprogress of the reaction was monitored by HPLC analysis and massspectroscopy. At completion of the reaction, the volatiles were removedin vacuo and the residue was washed with ethyl acetate (3×1.0 mL) toremove the unreacted bis-NHS ester. The residue was dried, re-dissolvedin anhydrous DMF (0.5 mL) and treated with a solution of peptide B andDIEA (2 equiv) in DMF (0.5 mL) for 24 h. The mixture was diluted withwater (1:1, v/v) and applied directly to a YMC C-18 reverse phase HPLCcolumn and purified by elution with a linear gradient of acetonitrileinto water (both containing 0.1% TFA). Fractions were analyzed byanalytical HPLC and those containing the pure product were combined andlyophilized to obtain the required dimer. The following dimers wereprepared by this method (structure, name, compound reference number):

For the preparation of the dimer D5, after the coupling reaction of theindividual peptides, 50 μl of hydrazine was added to the reactionmixture (to expose the lysine N^(ε)-amino group) and the solution wasstirred for 2 min. The reaction mixture was diluted with water (1.0 mL)and the pH was adjusted to 2 with TFA. This was then purified by themethod described above.

Synthesis of D27

Synthesis of 1 and 3

Synthesis of the monomers were carried out as described in Method 5 on a0.25 mmol scale employing as the starting resinFmoc-GGGK(iV-Dde)NH-PAL-PEG-PS resin. The peptide resin was washed anddried before cleavage or further derivatization by automated or manualmethods.

Procedure for Synthesis of 2 and 4

Appendage of Biotin-JJ, Lysyl, Glycyl and Serinyl(GalNAc(Ac)₃-α-Dmoieties onto 1 and 3 was done by manual SPPS such as described inMethod 6 and Method 8. The coupling of amino acids was carried out inDMF using HOBt/DIC activation (except for Ser(GalNAc(Ac)₃-α-D). Fmocremoval was carried out with 20% piperidine in DMF. All couplings were5-16 hours duration. After each coupling, the completion was confirmedby the Kaiser test. In the case of Ser(GalNAc(Ac)₃-α-D, the coupling wasperformed in DMF employing HATU/DIEA as the coupling agent. In caseswhere the Kaiser test indicated unreacted amino groups the coupling wasrepeated. Removal of the N-terminal Fmoc group and cleavage from resinwas performed. The crude peptide was precipitated in ether and washedtwice by ether and dried under vacuum. The linear crude peptide wasdirectly cyclized by dissolving the peptide in DMSO (40 mg/mL). The pHof the solution was adjusted to 8 by addition of aqueousN-methylglucamine and the solution was stirred in air for 48 h at roomtemperature. The peptides were then purified employing gradient HPLC asdescribed in Method 1 employing a Waters-YMC C-18 ODS preparative column(250 mm×4.6 mm i.d.). The pure product-containing fractions werecombined and lyophilized to provide the needed peptides.

Procedure: Synthesis of D27—Compound 6

To a solution of glutaric acid bis-NHS ester (0.122 mmol, PierceScientific Co.) in anhydrous DMF was added dropwise a solution of 4 inDMF (40 mg, 0.0122 mmol). DIEA was added to neutralize thetrifluoroacetic acid bound to the peptide and N-hydroxysuccinimideformed during the reaction. This 0.7 mL solution was stirred for 4 h.The reaction was monitored by HPLC and mass spectroscopy. DMF wasremoved under vacuum. The excess diester was removed by addition ofethyl acetate which precipitated the peptide-monoester 5 whiledissolving glutaric acid bis-NHS ester. The mixture was centrifuged andthe liquid portion decanted. This was repeated twice. The residue waskept under vacuum for 10 min. The residue was dissolved in DMF and mixedwith a solution of 2 (37 mg, 0.009 mmol) in DMF (pH 7). It was stirredat ambient temperature for 16 h. The volatiles were removed under highvacuum and the acetate functions were removed by treatment of theresidue with 1 mL of hydrazine/MeOH (15/85, v/v) solution with stirringfor 2.5 h at ambient temperature. Acetone was added to quench the excessof hydrazine and the volatiles were removed under vacuum. The resultingresidue was dissolved in DMSO and purified by preparative HPLC asdescribed above to provide 9 mg of the pure material.

Analytical Data

The HPLC analysis data and mass spectral data for the dimeric peptidesD1-5 and D8-30 identified above, as well as that for peptide componentsof dimer D27, are given in Table 6 below.

TABLE 6 Analytical Data for Homodimeric and Heterodimeric PeptideConstructs Retention Compound # Time (System) Mass Spectral data(API-ES, −ion) D1 8.98 min. (A) 1987.7 (M − 3H)/3, 1490.6 (M − 4H)/4,1192.3 (M − 5H)/5 D2 16.17 min (B) 2035.3 (M − 3H)/3, 1526.1 (M − 4H)/4,1220.7 (M − 5H)/5 D3 8.74 min (C) 1933.6 (M − 3H)/3, 1449.9 (M − 4H)/4,1159.4 (M − 5H)/5 D4 10.96 min (D) 2032.8 (M − 3H)/3 D5 6.57 min (E)1816.2 (M − 3H)/3, 1361.8 (M − 4H)/4, 1089.4 (M − 5H)/5, 907.7 (M −6H)/6 D8 4.96 min; (F) 2379.3 [M − 3H]/3 D9 5.49 min; (G) 2146.4 [M −3H]/3 D10 5.44 min; (H) 2082.7 [M − 3H]/3, 1561.7 [M − 4H]/4, 1249.1 [M− 5H]/5, 1040.7 [M − 6H]/6 D11 7.23 min; (E) 2041.8 [M − 3H]/3, 1531.1[M − 4H]/4, 1224.6 [M − 5H]/5 D12 5.84 min; (H) 1877.1 [M − 3H]/3,1407.6 [M − 4H]/4, 1125.9 [M − 5H]/5, 938.1 [M − 6H]/6. D13 5.367 min;(E) 1965.3 [M − 3H]/3, 1473.8 [M − 4H]/4, 1178.8 [M − 5H]/5, 982.2 [M −6H]/6 D14 4.78 min; (I) 2275.0 [M − 3H]/3, 1362.8 [M − 5H]/5 D15 5.41min; (H) 1561.3 [M − 4H]/4, 1249.1 [M − 5H]/5, 1040.8 [M − 6H]/6, 891.8[M − 7H]/7. D16 5.44 min; (J) 2150.8 [M − 3H]/3, 1613.1 [M − 4H]/4,1289.9 [M − 5H]/5, 1074.8 [M − 6H]/6, 920.9 [M − 7H]/7. D17 4.78 min;(K) 1789.4 [M − 3H]/3, 1347.7 [M − 4H]/4. D18 4.74 min; (L) 2083.1 [M −3H]/3, 1562.7 [M − 4H]/4, 1249.5 [M − 5H]/5. D19 7.13 min; (O) 1891.9 [M− 3H]/3, 1418.4 [M − 4H]/4, 1134.8 [M − 5H]/5, 945.5 [M − 6H]/6. D20 9.7min; (P) 2700.4 [M − 2H]/2, 1799.3 [M − 3H]/3 D21 6.1 min; (P) 2891.3 [M− 2H]/2, 1927.2 [M − 3H]/3, 1445.1 [M − 4H]/4, 1155.8 [M − 5H]/5. D226.23 min; (Q) 1994.4 [M − 3H]/3, 1495.7 [M − 4H]/4, 1196.3 [M − 5H]/5D23 7.58 min; (J) 1854.4 [M − 3H]/3, 1390.8 [M − 4H]/4, 1112.7 [M −5H]/5, 927 [M − 6H]/6 D24 8.913 min; (R) 1952.1 [M − 3H]/3, 1463.4 [M −4H]/4, 1171.1 [M − 5H]/5, 975.3 [M − 6H]/6 D25 5.95 min; (E) 1954.9 [M −3H]/3, 1466.1 [M − 4H]/4, 1172.4 [M − 5H]/5, 976.8 [M − 6H]/6. D26 6.957min; (S) 1759.1 [M − 3H]/3, 1319.6 [M − 4H]/4, 1055.1 [M − 5H]/5 D275.50 min; (M) 2317.6 [M − 3H]/3, 1737.2 [M − 4H]/4, 1389.3 [M − 5H]/5,1157.7 [M − 6H]/6. D28 4.89 min; (N) 6229 [M + H] D29 5.01 min; (N)2258.1 [M − 3H + TFA]/3 D30 4.35 min; (N) 2176.0 [M − 3H]/3, 1631.5 [M −4H]/4, 1302.6 [M − 5H]/5, 1087.7 [M − 6H]/6, 932.1 [M − 7H]/7 P12-Q 7.4min (T) 2041.3 [M − 2H]/2 P6-F-Q 8.0 min (T) 1636.3 [M − 2H]/2

HPLC Analysis Systems

System A: Column: YMC C-4 (4.6×250 mm); Eluents: A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA); Elution: Initial condition, 25% B, LinearGradient 25-60% B in 10 min; Flow rate: 2.0 ml/min; Detection: UV @ 220nm.

System B: Column: YMC C-4 (4.6×250 mm); Eluents: A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA); Elution: Initial condition, 25% B, LinearGradient 25-60% B in 20 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System C: Column: YMC C-4 (4.6×250 mm); Eluents: A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA); Elution: Initial condition, 30% B, LinearGradient 30-60% B in 10 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System D: Column: YMC C-4 (4.6×250 mm); Eluents: A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA); Elution: Initial condition, 20% B, LinearGradient 20-60% B in 10 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System E: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 10% B, LinearGradient 10-60% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System F: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 30% B, LinearGradient 30-70% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System G: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 30% B, LinearGradient 30-75% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System H: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 20% B, LinearGradient 20-52% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System I: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 10% B, LinearGradient 10-65% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System J: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 20% B, LinearGradient 20-60% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System K: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 5% B, LinearGradient 5-60% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System L: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 5% B, LinearGradient 5-65% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System M: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 15% B, LinearGradient 15-50% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System N: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 10% B, LinearGradient 20-80% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System O: Column: YMC-C18, 4.6×250 mm; Eluents:A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA): Elution: Initial condition, 30% B, LinearGradient 30-60% B in 10 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System P: Column: YMC-C18, 4.6×250 mm; Eluents:A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA): Elution: Initial condition, 20% B, LinearGradient 20-80% B in 20 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System Q: Column: YMC-C18, 4.6×250 mm; Eluents:A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA): Elution: Initial condition, 20% B, LinearGradient 20-60% B in 6 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System R: Column: YMC-C18, 4.6×250 mm; Eluents:A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA): Elution: Initial condition, 25% B, LinearGradient 25-60% B in 10 min; Flow rate: 2.0 mL/min; Detection: UV @ 220nm.

System S: Column: YMC-C18, 4.6×100 mm; Eluents:A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA): Elution: Initial condition, 10% B, LinearGradient 10-60% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

System T: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA),B: Acetonitrile (0.1% TFA): Elution: Initial condition, 15% B, LinearGradient 15-50% B in 8 min; Flow rate: 3.0 mL/min; Detection: UV @ 220nm.

Example 10 Competition with ¹²⁵I-VEGF for Binding to KDR on HUVECs andKDR-Transfected Cells

The following experiment assessed the ability of KDR-bindingpolypeptides, homodimers and heterodimers of the invention to competewith ¹²⁵I-labeled VEGF for binding to KDR expressed by transfected 293Hcells.

Protocol:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described herein. The cells were incubated with¹²⁵I-VEGF in the presence or absence of competing compounds (at 10 μM,0.3 μM, and 0.03 μM). After washing the cells, the bound radioactivitywas quantitated on a gamma counter. The percentage inhibition of VEGFbinding was calculated using the formula [(Y1-Y2)×100/Y1], where Y1 isspecific binding to KDR-transfected 293H cells in the absence peptides,and Y2 is specific binding to KDR-transfected 293H cells in the presenceof peptide competitors. Specific binding to KDR-transfected 293H cellswas calculated by subtracting the binding to mock-transfected 293H cellsfrom the binding to KDR-transfected 293H cells.

Results:

As shown in FIG. 14, all of the KDR-binding compounds assayed were ableto compete with ¹²⁵I-VEGF for binding to KDR-transfected cells. Theheterodimer (D1) was clearly the most effective at competing with¹²⁵I-VEGF, even over the two homodimers (D2 and D3), confirming thesuperior binding of D1.

Example 11 Receptor Activation Assay

The ability of KDR-binding multimeric constructs, includingheteromultimers of the invention, to inhibit VEGF induced activation(phosphorylation) of KDR was assessed using the following assay (seealso Example 4 above).

Protocol:

Dishes of nearly confluent HUVECs were placed in basal medium lackingserum or growth factors overnight. The next day, the dishes in group (c)below were pretreated for 15 min in basal medium with a KDR-bindingpeptide, then the cells in the dishes in groups (a), (b), and (c) wereplaced in fresh basal medium containing:

(a) no additives (negative control),

(b) 5 ng/mL VEGF (positive control), or

(c) 5 ng/mL VEGF plus the putative competing/inhibiting peptide.

After 5 min of treatment, lysates were prepared from the dishes.Immunoprecipitated KDR from the lysates was analyzed sequentially byimmunoblotting for phosphorylation with an anti-phosphotyrosineantibody, and for total KDR with an anti-KDR antibody (to control forsample loading).

Results:

As shown in FIG. 15, D1 was able to completely block the VEGF-inducedphosphorylation of KDR in HUVECs at 10 nM. More than half of thephosphorylation was inhibited by the compound at 1 nM. Homodimers D2 andD3, made up of the two individual binding moieties that are contained inD1, had no effect on phosphorylation at up to 100 nM, demonstrating thebenefit achievable by using an appropriate heterodimer to block areceptor-ligand interaction. In multiple experiments, the IC₅₀ for D1 inthis assay varied between 0.5 and 1 nM. A different heterodimercontaining unrelated binding sequences, D31 (structure shown below), hadno effect on phosphorylation at 100 nM in spite of it's high bindingaffinity (11 nM for KDR by Biacore), suggesting that the choice ofKDR-binding moieties is important when constructing a multimer tocompete with VEGF for binding to KDR. Even though the affinity of D1 forKDR is 10-fold higher than that of D2 (by SPR analysis), it's IC₅₀ inthe activation assay is at least 100-fold lower, suggesting thattargeting two distinct epitopes on KDR with a single binding moleculecan generate greater steric hindrance than a molecule with similaraffinity that only binds to a single epitope on KDR. Similarly, itshould be pointed out that the two KDR-binding moieties within D1 whentested as monomeric free peptides (P12-XB and P6-D) in the receptoractivation assay had IC₅₀ s of 0.1 and 1 micromolar respectively, whichis 100 to 1000-fold higher than the IC₅₀ for D1 in the assay and 14 to30-fold higher than the K_(D)s for the fluoresceinated derivatives ofthe monomeric peptides. Thus, creating a dimer containing two peptideswith weak VEGF-blocking activity has resulted in a molecule with verypotent VEGF-blocking activity that goes well beyond the increasedbinding affinity of D1.

Example 12 Migration Assay

The following experiment assessed the ability of D1, a heteromultimer ofthe invention, to block the VEGF-induced migration of HUVECs in culture.

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD Matrigel-coated FluoroBlok 24-well insert plates(#354141). Basal medium, containing either nothing or differentattractants such as VEGF (10 ng/mL) or serum (5% FBS) in the presence orabsence of potential VEGF-blocking/inhibiting compounds, was added tothe lower chamber of the wells. After 22 hours, quantitation of cellmigration/invasion was achieved by post-labeling cells in the insertplates with a fluorescent dye and measuring the fluorescence of theinvading/migrating cells in a fluorescent plate reader. The VEGF-inducedmigration was calculated by subtracting the migration that occurred whenonly basal medium was placed in the lower chamber of the wells.

Results:

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by D1. At 5 nM D1, the VEGF-stimulatedendothelial cell migration was 84% blocked (see FIG. 16). At 25 nM D1,this migration was almost completely blocked. In other experiments, aknown KDR inhibitor, SU-1498((E)-3-(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)aminocarbonyl]acrylonitrile]was tested in the assay. SU1498 at 3 micromolar did not block theVEGF-induced migration as well as D1 (47% blocked at 3 micromolar). D7(structure provided below), at 50 nM, also produced essentially completeinhibition of the migration stimulated by VEGF. Serum was a verypowerful attractant in the assay when used in place of VEGF, but itseffect was not significantly diminished by D1, indicating that D1specifically inhibits endothelial migration induced by VEGF.

Example 13 The Following Experiments Describe Methods used to PrepareTc, In, Lu, and I-Labelled Compounds Preparation of ^(99m)Tc-P12-P

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA.2.5H₂O (Fluka) in 1 mL of water. The pHof the stannous DTPA solution was adjusted to pH 6-8 using 1N NaOH. 50μg of P12-P(Ac-AGPTWC*EDDWYYC*WLFGTGGGK(PnAO6—NH-(0=)C(CH₂)₃C(═O)-JJ)-NH₂) in 50 μLof 10% DMF was mixed with 20 μL of ^(99m)TcO₄ ⁻ (2.4 to 4 mCi, Syncor),followed by 100 μL of the stannous Sn-DTPA solution. After 30 minutes atRT, the radiochemical purity (RCP) was 93%. The product was purified ona Supelco Discovery C16 amide column (4×250 mm, 5 um pore size) elutedat a flow rate of 0.5 mL/min using an aqueous/organic gradient of 1 g/Lammonium acetate in water (A) and acetonitrile (B). The followinggradient was used: 30.5% B to 35% B in 30 minutes, ramp up to 70% B in10 min. The compound, which eluted at a retention time of 21.2 minuteswas collected into 500 μL, of 50 mM citrate buffer (pH 5.2) containing1% ascorbic acid and 0.1% HSA, and acetonitrile was removed using aSpeed Vacuum (Savant). After purification, the compound had an RCP of>98%.

Preparation of ¹¹¹In-P12-XDT

50 μg of P12-XDT (Ac-AGPTWCEDDWYYCWLFGTJK(JJ-DOTA)-NH₂) in 50 μL, of 10%DMF was mixed with ¹¹¹InCl₃ (50 μL, 400 μCi, Mallinckrodt) and 100 μL,of 0.2M ammonium acetate or citrate buffer at a pH of 5.3. After beingheated at 85° C. for 45 minutes, the radiochemical purity (RCP) rangedfrom 44% to 52.2% as determined using HPLC. The ¹¹¹In-labeled compoundwas separated from unlabeled ligand using a Vydac C18 column (4.6×25 cm,5 micron pore size) under the following conditions: aqueous phase, 1 g/Lammonium acetate (pH 6.8); organic phase, acetonitrile. Gradient: 23%org. to 25% org. in 30 minutes, up to 30% org. in 2 minutes, hold for 10minutes. The compound, which eluted at a retention time of 20.8 min, wascollected into 200 μL, of 50 mM citrate buffer (pH 5.2) containing 1%ascorbic acid and 0.1% HSA, and the acetonitrile was removed using aSpeed Vacuum (Savant). After purification the compound had an RCP of>93%.

Preparation of ¹¹¹In-D4

A histidine buffer was prepared by adjusting a 0.1M solution ofhistidine (Sigma) to pH 6.25 with concentrated ammonium hydroxide.Ammonium acetate buffer was prepared by adjusting a 0.2 M solution ofammonium acetate (99.99%, Aldrich) to pH 5.5 using concentrated HCl (J.T. Baker, Ultra Pure). High purity ¹¹¹InCl₃ (100 μL, 1.2 mCi,Mallinckrodt) was added to D4 (200 μg in 200 of 50% DMF, 10% DMSO, 20%acetonitrile and 20% water), followed by addition of 300 μL, ofhistidine buffer. The final pH was 5.5. After incubation of the reactionmixture at 85° C. for 45 minutes, the RCP was 20%.

Alternatively, ¹¹¹InCl₃ provided with a commercially availableOctreoScan™ Kit (134 μL, 0.6 mCi, Mallinkrodt) was added to D4 (135 μg)in 162 μL of 0.2M ammonium acetate buffer. The final pH was 5.5. Afterincubation of the reaction mixture at 85° C. for 45 min. the RCP was20%.

Preparation of ¹²⁵I-D5

D5 (200 μg), in 30 μL of DMF that had been previously adjusted to pH8.5-9.0 using diisopropyl amine, was added to 1 mCi of mono-iodinated¹²⁵I Bolton-Hunter Reagent (NEX-120, Perkin-Elmer) that had beenevaporated to dryness. The vial was shaken and then incubated on ice for30 minutes with occasional shaking. After this time, the RCP was 23%.¹²⁵I-D5 (shown below) was purified by HPLC at a flow rate of 1 mL/minusing a Vydac C18 column (4.6×250 mm, 5 micron pore size) under thefollowing conditions. Aqueous phase: 0.1% TFA in water; organic phase:0.085% TFA in acetonitrile. Gradient: 30% org. to 36% org. in 30minutes, up to 60% org. in 5 minutes, hold for 5 minutes. The compoundwas collected into 200 μL of 50 mM citrate buffer (pH 5.2) containing 1%ascorbic acid and 0.1% HSA. Acetonitrile was removed using a SpeedVacuum (Savant). The resulting compound had an RCP of 97%.

Preparation of ¹⁷⁷Lu-D11

D11 (5 μL of a ˜1 μg/μL solution in 0.05N NH₄OH/10% EtOH) was added to aglass insert microvial containing 80 μL of 0.2M NaOAc buffer, pH 5.6.Enough ¹⁷⁷Lu was added to bring the ligand:Lu ratio to ≦2:1 (1-5 mCi).The vial was crimp-sealed and heated at 100° C. for 15-20 minutes,cooled for 5 minutes, and treated with 3 μL of 1% Na₂EDTA.2H₂O in H₂O.The entire reaction mixture was injected onto a Supelco Discovery RPAmide C16 column (4 mm×250 mm×5 μm). The following HPLC conditions wereused: Column temperature=50° C., Solvent A=H₂O w/0.1% TFA, Solvent B=ACNw/0.085% TFA, gradient 0.6/0.25 mL/min A/B at t=0 minutes to 0.5/0.4mL/min A/B at t=60 minutes. The retention time for D11 was ˜40 minutes;that of ¹⁷⁷Lu-D11 1334 was ˜42 minutes. The radioactive peak wascollected into 0.7 ml of 0.05M citrate buffer, pH 5.3 containing 0.1%Human Serum Albumin Fraction V and 1.0% Ascorbic Acid, and the mixturewas spun down in a Savant Speed Vac to remove organic solvents.Radiochemical purities of greater than 80% were obtained.

Preparation of ^(99m)Tc-D12

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA.2.5H₂O (Fluka) in 1 mL of water. D12 (100μg in 100 μL of 50% DMF) was mixed with 75 μL of 0.1 M, pH 9 phosphatebuffer and 60 μL of ^(99m)TcO₄ ⁻ (2.4 to 4 mCi, Syncor), followed by 100μL of the stannous Sn-DTPA solution. After 10 min at 40° C., theradiochemical purity (RCP) was 16%. The product was purified on aSupelco Discovery C16 amide column (4×250 mm, 5 um pore size) eluted ata flow rate of 0.7 mL/min using an aqueous/organic gradient of 0.1% TFAin water (A) and 0.085% TFA in acetonitrile (B). The following gradientwas used: 30% B to 42% B in 36 min, ramp up to 70% B in 10 min. Thecompound, which eluted at a retention time of 37.1 min. was collectedinto 500 μL of 50 mM citrate buffer (pH 5.2) containing 0.2% HSA, andacetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of >90%.

Preparation of ^(99m)Tc-D14

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA.2.5H₂O (Fluka) in 1 mL of water. D14 (100μg in 100 μL of 50% DMF) was mixed with 50 μL of ^(99m)TcO₄ ⁻ (6 mCi,Syncor) and 125 μL of 0.1M phosphate buffer, pH 9 followed by 100 μL ofthe stannous Sn-DTPA solution. After 15 min at 40°, the radiochemicalpurity (RCP) was 21%. The product was purified on a Vydac peptide C18column (4.6×250 mm) eluted at a flow rate of 1 mL/min using anaqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA inacetonitrile (B). The following gradient was used: 30% B to 45% B in 40min. The compound, which eluted at a retention time of 34.9 min., wascollected into 500 μL of 50 mM citrate buffer (pH 5.3) containing 0.2%HSA, and acetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of 92.5%.

Example 14 Binding of ¹²⁵I-Labeled Heteromultimers of the Invention toKDR-Transfected Cells

An experiment was performed to test the ability of ¹²⁵I-labeled D5 tobind to KDR-transfected 293H cells. In this experiment, differentamounts of ¹²⁵I-labeled D5 (1-4 μCi/ml, labeled with ¹²⁵I-Bolton-Hunterreagent and HPLC-purified) were incubated with mock & KDR-transfected293H cells in 96-well plates for 1 hr at room temperature. Binding wasperformed with and without 40% mouse serum to evaluate the serum effecton binding to KDR-transfected cells. After washing away the unboundcompound, the cells in each well were lysed with 0.5 N NaOH and thelysates were counted with a gamma counter.

The results of this experiment are summarized in FIG. 17 and FIG. 18. Itis clearly evident from these results that ¹²⁵I-labeled D5 is able tospecifically bind to KDR-transfected cells and its binding is notaffected by the presence of 40% mouse serum. Somewhat more binding toKDR-transfected cells was observed in the absence of serum as comparedto binding in the presence of 40% mouse serum. However, the binding of¹²⁵I-D5 to mock-transfected cells was also increased by about the sameextent when serum was omitted during the assay, indicating that theincreased binding in the absence of serum was non-specific (FIG. 17).Specific binding to KDR-transfected cells (after subtracting binding tomock-transfected cells) looked almost identical with or without mouseserum (as shown in FIG. 18). In this experiment, 10-14% of the total CPMadded were specifically bound to KDR-transfected cells (data not shown).

Example 15

A peptide heterodimer (D6, shown below) was prepared as previouslydescribed in Example 9 using glutaric acid bis N-hydroxysuccinimidylester. The heterodimer was tested for binding to KDR-Fc using Biacoreand an affinity contant was determined as follows.

Three densities of KDR-Fc were cross-linked to the dextran surface of aCM5 sensor chip by the standard amine coupling procedure (0.5 mg/mLsolution diluted 1:100 or 1:50 with 50 mM acetate, pH 6.0). Flow cell 1was activated and then blocked to serve as a reference subtraction.Final immobilization levels achieved:

R_(L) Fc 2 KDR-Fc=1607

R_(L) Fc 3 KDR-Fc=3001

R_(L) Fc 4 KDR-Fc=6319

Experiments were performed in PBS buffer (5.5 mM phosphate, pH 7.65,0.15 M NaCl)+0.005% P-20 (v/v)). D6 was diluted to 250 nM in PBS andserial dilutions were performed to produce 125, 62.5, 31.3 15.6, 7.8,and 3.9 nM solutions. All samples were injected in duplicate. Forassociation, peptides were injected at 20 μL/min for 12.5 minutes usingthe kinject program. Following a 10 minute dissociation, any remainingpeptide was stripped from the KDR surface with a quickinject of 50 mMNaOH+1M NaCl for 12 s at 75 μL/min. Sensorgrams were analyzed usingBIAevaluation software 3.1 and a hyperbolic double rectangularregression equation in SigmaPlot 6.0. Heterodimer steady state bindingaffinities (KD_(AV)) were determined at all three KDR immobilizationdensities (Table 7).

TABLE 7 Summary of Parameters KD₁ (nM) RMax₁ KD_(AV) (nM) RMax_(AV) R²*D6 vs. 1600RU 46 13.1 1.5 12.6 0.995 vs. 3000RU 25.5 21.2 0.665 22.70.991 vs. 6000RU 17 61.3 0.662 62.2 0.993From this data, it appears that at the higher immobilization densities,the heterodimer binds KDR with a sub-nanomolar affinity (˜0.6 nM).

To assess the in-vivo clearance of this peptide heterodimer, a smallamount of material was iodinated using iodogen and Na¹²⁵I according tostandard protocols (Pierce). Radio iodination was done in the RadiationSafety lab, within the designated hood. One tube coated with the iodogenreagent was pre-wet with 1 mL of 25 mM Tris, 0.4M NaCl, pH 7.5. This wasdiscarded and 100 μl of the same buffer added. Using a Hamilton syringe11 μL of the ¹²⁵I-NaI was transferred to the reaction tube. Based onoriginal estimates of the Na¹²⁵I concentration of 143.555 mCi/ml, the 11μL should contain about 1.5 mCi. No dose calibrator was in the room.After addition, the sample was swirled and set in a lead pig to incubatefor 6 min with a swirl every 30 sec. After 6 min, the entire sample wastransferred to the peptide that was in an Eppendorf tube. The sample wasswirled and set to incubate for 8 min, with a swirl every 30 sec. After8 min the reaction was quenched (terminated) with tyrosine (10 mg/mL, asaturated solution), allowed to sit for 5 min, and then 2 μL was removedfor a standard.

For purification a 10 mL column of the D-salt polyacrylamide 1800 wasused to separate the labeled peptide from labeled tyrosine. The columnwas first washed with 10 mL saline, then 5 mL of 25 mM Tris, 0.4M NaCl,pH 7.5 containing 2.5% HSA to block non-specific sites. After the HSAbuffer wash, the column was eluted with 60 mL of the mM Tris, 0.4 M NaClbuffer, and the column was stored overnight at 4° C. The labeled samplecontained 1.355 mCi, as determined by the dose calibrator. The 2 μlsample that was removed as a standard contained 8.8 μCi. The peptidesample was applied to the D-salt 1800 column and eluted with theTris/NaCl buffer, pH 7.5. The flow was controlled by applying single 0.5ml aliquots for each fraction, #1-14, and then 1.0 mL for fractions25-43. The peak of activity in fractions # 9, 10, and 11, was assumed tobe the peptide. The radioactivity in 24 through ˜40 is likely thelabeled tyrosine. From this purification, fractions #9-12 were pooledtogether and used for the subsequent clearance study (concentration of¹²⁵I-D6 in pool is 7.023 μg/mL; 100 μL=0.702 μg with 8.6 μCi).

A total of 15 mice were injected with 100 μL ¹²⁵I-D6 and mice (in setsof 3) were sacrificed at the following time points: 0, 7, 15, 30, 90minutes. Actual activity injected was about 6 μCi. With 6 μCi injectedthe corresponding peptide administered was ˜0.5 μg per animal. Oncesacrificed, the counts were determined in a 50 μL plasma sample fromeach animal. For each set of three animals at each time point, thecounts were averaged, converted to % injected dose/ml plasma (ID %/mL),and then plotted to assess the rate of clearance (FIG. 19). Then thisdata was fit to either a 4 or 5 parameter equation to determine thebiphasic half life of this molecule. The 4 parameter fit resulted in aT_(1/2α) of 2.55 minutes and a T_(1/2β) of 64.66 minutes. The 5parameter fit resulted in a T_(1/2α) of 2.13 minutes and a T_(1/2β) of23.26 minutes.

Besides taking counts from the plasma samples, larger volumes of plasmawere taken from mice sacrificed at the 0, 30, and 90 minute time points.These samples were injected onto a Superdex peptide column (Pharmacia)coupled to a radioactivity detector to assess the association of thepeptide with serum proteins (FIG. 20). As shown, the labeled peptidedoes associate with higher MW proteins, which could explain its biphasichalf life clearance behavior.

To help assess the potency of the peptide as an anti-angiogenesisinhibitor, D6 was tested in an endothelial cell proliferation assayusing HUVECs and BrdU detection. Briefly, freshly isolated HUVECs(between p3-6) were cultured in RPMI+10% FCS+1% antibiotics+1%1-glutamin+0.4% BBE (bovine brain extract) and seeded per well,5000-10000/well in 100 μL. The cells were allowed to recover for 24 hprior to use. Then the cells were washed with PBS twice and treated for48 h with anti-VEGF antibody (positive control) or peptides A, B and C(0.1 and 10 ug/mL) in RPMI+0.1% BSA+1% 1-glutamin. The following 6variables were tested in 2 series (n=4):

Series I: w/o VEGF

Series II: w/ VEGF (30 ng/mL)

-   -   1. Standard medium: RPMI+10% FCS+1% antibiotics+1%        1-glutamin+0.4% BBE    -   2. Negative control 1: RPMI (true starvation)    -   3. Negative control 2: RPMI+0.1% BSA+1% 1-glutamin    -   4. Positive control: anti-VEGF 10 μg/ml in RPMI+0.1% BSA+1%        1-glutamin    -   5. 0.1 μg/ml KDR peptides in RPMI+0.1% BSA+1% 1-glutamin    -   6. 10 μg/ml KDR peptides in RPMI+0.1% BSA+1% 1-glutamin

Protocol:

1) cells are incubated for 48 hours under various conditions

2) 10 μL BrdU dilution (1:100 in EBM) is added to each well at 24 hours

3) incubate for another 24 hours (total 48 hrs)

4) aspirate the culture medium

5) add 100 μL FixDenat to each well, incubate at room temperature for 30min.

6) Discard FixDenat solution

7) 100 μL antibody-solution (PBS 1% BSA and anti-BrdU PO) added to eachwell.

8) incubate at RT for 90 minutes.

9) wash 3 times with PBS, 2004/well, 5 min.

10) add substrate solution (TMB), incubate for 10-30 minutes

11) transfer all to a flexible plate

12) stop the reaction by adding 2M H₂SO₄, 25 μL/well

13) read absorbance at 450 nm within 5 minutes after stopping thereaction.

Note: Background binding was determined by omitting the anti-BrdUantibody in 4 wells with control cells (cultured in complete medium;EBM+BulletKit) and by complete labeling of cells that was not exposed toBrdU.

Of the two KDR binding constructs tested (D6 and P12-G(Ac-AGPTWC*EDDWYYC*WLFGT-GGGK-NH₂)) as shown in FIG. 21, D6 completelyinhibits HUVEC proliferation at 10 μg/mL in the presence of VEGF,similar to an anti-VEGF antibody (positive control). PNC-1(Ac-AEGTGDLHCYFPWVCSLDPGPEGGGK-OH) (SEQ ID NO: 29) was used as negativecontrol. On the other hand, P12-G (one of the peptides that make up theheterodimer) does not inhibit proliferation this assay at the highestconcentration tested (10 μg/mL). As a result, the heterodimer clearlyshows an enhanced ability to compete with VEGF in comparison with P12-Galone.

Example 16 BIAcore Analysis Murine KDR-Fc Binding of Peptide Dimers D1and D7

Using BIAcore, determine the binding constants of peptide dimers D1 (aheterodimer of P12-G and a truncated form of P6-D) and D7 (a heterodimerof P5-D and P6-D) for murine KDR-Fc.

Procedure

Three densities of recombinant murine KDR-Fc were cross-linked to thedextran surface of a CM5 sensor chip by the standard amine couplingprocedure (0.5 mg/mL solution diluted 1:100 or 1:40 with 50 mM acetate,pH 6.0). Flow cell 1 was activated and then blocked to serve as areference subtraction. Final immobilization levels achieved:

R_(L) Fc 2 KDR-Fc=2770 R_(L) Fc 3 KDR-Fc=5085 R_(L) Fc 4 KDR-Fc=9265

Experiments were performed in PBS buffer (5.5 mM phosphate, pH 7.65,0.15 M NaCl)+0.005% P-20 (v/v)). P12-G, run as a control, was diluted to125 nM in PBS. Serial dilutions were performed to produce 62.5, 31.3,15.6, 7.8, and 3.9 nM solutions. D1 and D7 were diluted to 50 nM in PBSand serial dilutions were performed to produce 25, 12.5, 6.25, 3.13,1.56, 0.78, and 0.39 nM solutions. All samples were injected induplicate. For association, peptides were injected at 30 μL/min for 3minutes using the kinject program. Following a 10 minute dissociation,any remaining peptide was stripped from the rmKDR-Fc surface with aquickinject of 50 mM NaOH+1M NaCl for 12 s at 75 μL/min.

Sensorgrams were analyzed using the simultaneous ka/kd fitting programin the BIAevaluation software 3.1. The Results are shown in Table 8 andFIGS. 22-24. The fact that about the same K_(D2) constant was achievedfor both heterodimers even when the density of receptor on the sensorchip was reduced by half is consistent with multimeric binding of theheterodimers to individual receptors rather than cross-link-type bindingbetween receptors.

TABLE 8 Summary of Kinetic Parameters. ka1 (1/Ms) kd1 (1/s) ka2 (1/RUs)kd2 (1/s) KD1^(#) (nM) KD2^(‡) (nM) Chi²* D1 vs. 2700RU 7.94E+05 0.01393.31E−04 5.96E−04 17.5 0.751 0.077 vs. 5000RU 5.54E+05 8.88E−03 1.17E−044.57E−04 16.0 0.825 0.323 D7 vs. 2700RU 7.59E+05 0.011 3.36E−04 6.44E−0414.5 0.848 0.082 vs. 5000RU 5.21E+05 7.39E−03 1.17E−04 4.68E−04 14.20.898 0.278 Fluorescein vs. 2700RU 1.02E+06 0.037 — — 36.4 — 0.073labeled P12-G vs. 5000RU 5.18E+05 0.0174 — — 33.6 — 0.167 ^(#)K_(D1) isa calculated K_(D) based on kd₁/ka₁ ^(‡)K_(D2) is a calculated K_(D)based on kd₂/ka₁ (i.e. avidity factor) *The chi2 value is a standardstatistical measure of the closeness of the fit. For good fitting toideal data, chi2 is of the same order of magnitude as the instrumentnoise in RU (typically <2).

Example 17 Demonstration of the Distinction Between Binding Affinity andBiological Potency Through In Vitro Assays

The following experiments showed that heteromultimers can display muchgreater biological potency than a monomeric peptide with similar bindingaffinity to the same target.

Protocol Experiment 1:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described in Example 6. The cells were incubatedwith ¹²⁵I-VEGF in the presence or absence of PG-1(Ac-ERVTTCWPGEYGGVECYSVAY-NH₂) (SEQ ID NO: 30) or D1 (at 300, 30, 3, and0.3 nM). After washing the cells, the bound radioactivity wasquantitated on a gamma counter. The percentage inhibition of VEGFbinding was calculated using the formula [(Y1-Y2)×100/Y1], where Y1 isspecific binding to KDR-transfected 293H cells in the absence peptides,and Y2 is specific binding to KDR-transfected 293H cells in the presenceof peptide competitors. Specific binding to KDR-transfected 293H cellswas calculated by subtracting the binding to mock-transfected 293H cellsfrom the binding to KDR-transfected 293H cells.

Protocol Experiment 2:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, with or without VEGF (10 ng/mL) in the presence or absenceof increasing concentrations of PG-1 or D1, was added to the lowerchamber of the wells. After 22 hours, quantitation of cellmigration/invasion was achieved by post-labeling cells in the insertplates with a fluorescent dye and measuring the fluorescence of theinvading/migrating cells in a fluorescent plate reader. VEGF-stimulatedmigration was derived by subtracting the basal migration measured in theabsence of VEGF.

Results Experiment 1:

As shown in FIG. 26, PG-1 and D1 competed about equally well with¹²⁵I-VEGF for binding to KDR-transfected cells, indicating that theypossess comparable binding affinities as well as a comparable ability toinhibit VEGF from binding to KDR.

Results Experiment 2:

In spite of the fact that both PG-1 and D1 potently block ¹²⁵I-VEGFbinding to KDR-expressing cells to the same degree (FIG. 26), theheterodimeric D1 was significantly more potent in blocking thebiological effects of VEGF as demonstrated in an endothelial cellmigration assay (FIG. 27) than the monomeric PG-1. At up to 62.5 nM,PG-1 had no effect on VEGF-stimulated migration whereas D1 completelyblocked VEGF-stimulated migration at 50 nM. These data suggest thatheteromultimeric binding can more effectively block the biologicalactivity of a ligand than a monomer, even when the monomer has acomparable ability to inhibit ligand binding to its receptor.

Example 18 Binding of Tc-Labeled Heterodimers of the InventionKDR-Transfected 293H Cells

In this Example, the ability of Tc-labeled D10 to bind KDR was assessedusing KDR-transfected 293H cells. The results show that Tc-labeled D10bound significantly better to KDR transfected 293H cells than to mocktransfected 293H cells, and good binding was maintained in the presenceof 40% mouse serum. In addition, a derivative of Tc-labeled D10 with itsamino acid sequence scrambled, D18, was shown to possess no affinity forKDR-expressing cells, confirming the specificity of the D10 binding tothose cells.

Synthesis of ^(99m)Tc-Labeled Peptides Preparation of ^(99m)Tc-D10

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA.2.5H₂O (Fluka) in 1 mL of water. D10 (100μg in 100 μL of 50% DMF) was mixed with 75 μL of 0.1 M, pH 9 phosphatebuffer and 50 μL of ^(99m)TcO₄ ⁻ (2.4 to 5 mCi, Syncor), followed by 100μL of the stannous Sn-DTPA solution. After 15 min at RT, theradiochemical purity (RCP) was 72%. The product was purified on aSupelco Discovery C16 amide column (4×250 mm, 5 um pore size) eluted ata flow rate of 0.7 mL/min using an aqueous/organic gradient of 0.1% TFAin water (A) and 0.085% TFA in acetonitrile (B). The following gradientwas used: 30% B to 42% B in 36 min, ramp up to 70% B in 10 min. Thecompound, which eluted at a retention time of 32 min. was collected into500 μL of 50 mM citrate buffer (pH 5.2) containing 0.2% HSA, andacetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of >90%.

Preparation of ^(99m)Tc-D18

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA.2.5H₂O (Fluka) in 1 mL of water. D18 (100μg in 100 μL of 50% DMF) was mixed with 50 μL of 0.1 M, pH 9 phosphatebuffer and 90 μL of ^(99m)TcO₄ ⁻ (14 mCi, Syncor), followed by 100 μL ofthe stannous Sn-DTPA solution. The reaction was warmed for 20 minutes at37° C. The entire reaction was injected on a Vydac 218TP54 C18 column(4.6×250 mm, 5 um silica) and eluted at a flow rate of 1.5 mL/min usingan aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA inacetonitrile (B). The following gradient was used: 32% to 39% B in 30minutes, ramp up to 80% B in 2 min. The free ligand eluted at aretention time of 19 minutes. The complex, which eluted at 24 minutes,was collected into 500 μL of 50 mM citrate buffer (pH 5.3) containing0.1% HSA and 1% Ascorbic Acid. Acetonitrile & excess TFA were removedusing a Speed Vacuum (Savant) for 40 minutes. After purification, thecompound had an RCP of 93%.

Transfection of 293H Cells

293H cells were transfected using the protocol described in Example 6.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. # 354640). The cells in one half of the plate (48 wells) weremock-transfected (with no DNA) and the cells in the other half of theplate were transfected with KDR cDNA. The cells were 80-90% confluent atthe time of transfection and completely confluent the next day, at thetime of the assay; otherwise the assay was aborted.

Preparation of Opti-MEMI Media with 0.1% HSA

Opti-MEMI was obtained from Invitrogen (cat. # 11058-021) and humanserum albumin (HSA) was obtained from Sigma (cat. # A-3782). To prepareopti-MEMI media with 0.1% HSA, 0.1% w/v HSA was added to opti-MEMI,stirred at room temperature for 20 minutes, and then filter sterilizedusing 0.2 μM filter.

Preparation of Tc-Labeled Peptide Dilutions for the Assay

Stock solutions of Tc-labeled D10 and D18 were diluted in opti-MEMI with0.1% HSA to provide solutions with final concentrations of 1.25, 2.5,5.0, and 10 μCi/mL of each Tc-labeled heterodimer. A second set ofdilutions was also prepared using a mixture of 40% mouse serum/60%opti-MEMI with 0.1% HSA as the diluent.

Assay to Detect the Binding of the Tc-Labeled Heterodimers

Cells were used 24 h after transfection, and to prepare the cells forthe assay, they were washed 1× with 100 μL, of room temperatureopti-MEMI with 0.1% HSA. After washing, the opti-MEMI with 0.1% HSA wasremoved from the plate and replaced with 70 μL, of 1.25, 2.5, 5.0, and10 μCi/mL of Tc-labeled D10 or D18 (prepared as above with both diluentsolutions). Each dilution was added to three separate wells of mock andKDR-transfected cells. After incubating at room temperature for 1 h, theplates were washed 5 times with 100 μL, of cold binding buffer(opti-MEMI with 0.1% HSA). 100 μL, of solubilizing solution (0.5 N NaOH)was added to each well and the plates were incubated at 37° C. for 10minutes. The solubilizing solution in each well was mixed by pipeting upand down, and transferred to 1.2 mL tubes. Each well was washed oncewith 100 μL, of solubilizing solution and the washes were added to thecorresponding 1.2 mL tube. Each 1.2 mL tube was then transferred to a15.7 mm×100 cm tube to be counted in an LKB Gamma Counter.

Binding of Tc-Labeled Heterodimers to KDR Transfected Cells

The ability of Tc-labeled D10 and D18 to bind specifically to KDR wasdemonstrated using transiently transfected 293H cells. As shown in FIG.28A, Tc-labeled D10 bound significantly better to KDR transfected 293Hcells, as compared to mock-transfected 293H cells in both the presenceand absence of 40% mouse serum, although there was some inhibition inthe presence of serum. The total specific binding of this Tc-labeledheterodimer to KDR-expressing cells was much greater than that observedpreviously with a Tc-labeled monomeric peptide (Example 5). Tc-labeledD18, on the other hand, displayed no affinity for eithermock-transfected or KDR-transfected 293H cells, confirming thespecificity of D10 binding.

Example 19 Binding of a Lu-Labeled Heterodimers to KDR-Transfected 293HCells

In this Example, the ability of Lu-labeled D13 to bind KDR was assessedusing KDR-transfected 293H cells. The results show that Lu-labeled D13bound significantly better to KDR transfected 293H cells than to mocktransfected 293H cells, and good binding was maintained in the presenceof 40% mouse serum.

Synthesis of ¹⁷⁷Lu-Labeled Peptide Preparation of ¹⁷⁷Lu-D13

D13 (306 μg) was added to a 2-mL autosampler vial with a ˜450 μL conicalinsert and dissolved in 0.01N NH₄OH (50 μL). To this was added 300 μL of0.5M Ammonium Acetate containing stabilizers. A 6.8 μL aliquot of¹⁷⁷LuCl₃ in 0.05N HCl (39.3 mCi) was added, the vial was crimp-sealed,warmed for 15 min at 37° C., cooled for ˜5 minutes, and 10 μL of 1%Na₂EDTA.2H₂O in H₂O was added. A 350 μL aliquot of the reaction mixturewas injected onto a Supelco Discovery RP Amide C16 column (4 mm×250 mm×5μm). The following HPLC conditions were used: Column temperature=37° C.,Solvent A=H₂O containing 2 g/L NH₄OAc buffer, pH 7.0, Solvent B=80%ACN/20% H₂O, gradient 0.56/0.24 mL/min A/B at t=0 minutes to 0.47/0.33mL/min A/B at t=30 minutes. The retention time for D13 was ˜28 minutes;the retention time for ¹⁷⁷Lu-D13 was ˜29 minutes. The radioactive peakwas collected into 1 mL of a buffer containing stabilizers, final pH=7.6adjusted with Sodium Hydroxide. It was then spun down ˜40 minutes usinga Speed Vacuum (Savant) to remove ACN. The RCP of the isolated productwas 86%.

Transfection of 293H Cells

293H cells were transfected using the protocol described in Example 6.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. # 354640). The cells in one half of the plate (48 wells) weremock-transfected (with no DNA) and the cells in the other half of theplate were transfected with KDR cDNA. The cells were 80-90% confluent atthe time of transfection and completely confluent the next day, at thetime of the assay; otherwise the assay was aborted.

Preparation of Opti-MEMI Media with 0.1% HSA

Opti-MEMI media with 0.1% HAS was prepared as in Example 18.

Preparation of Lu-Labeled Peptide Dilutions for the Assay

A stock solution of Lu-labeled D13 was diluted in opti-MEMI with 0.1%HSA to provide solutions with final concentrations of 1.25, 2.5, 5.0,and 10 μCi/mL of labeled heterodimer. A second set of dilutions was alsoprepared using a mixture of 40% mouse serum/60% opti-MEMI with 0.1% HSAas the diluent.

Assay to Detect the Binding of the Lu-Labeled Heterodimers

This was carried out as detailed in Example 18 except that Lu-labeledD13 was used in place of the Tc-labeled heterodimers.

Binding of Lu-Labeled Heterodimer to KDR Transfected Cells

The ability of Lu-labeled D13 to bind specifically to KDR wasdemonstrated using transiently-transfected 293H cells. As shown in FIG.29, Lu-labeled D13 bound significantly better to KDR transfected 293Hcells, as compared to mock-transfected 293H cells in both the presenceand absence of 40% mouse serum, although there was some bindinginhibition in the presence of serum.

Example 20 Radiotherapy with a Lu-Labeled Heterodimers in Tumor-BearingMice

In this Example, the ability of Lu-labeled D13 to inhibit the growth ofPC3 cell tumors implanted in nude mice is demonstrated.

Synthesis of ¹⁷⁷Lu-Labeled D13 ¹⁷⁷Lu-Labeled D13 was Prepared asDescribed in Example 19

Animal Model

PC3 cells from ATCC, grown as recommended by the supplier, were injectedsubcutaneously between the shoulder blades of nude mice. When theirtumors reached 100-400 mm³, twelve mice were injected i.v. with 500microcuries of Lu-labeled D13 and their growth monitored for anadditional 18 days. Mice were sacrificed if they lost 20% or more oftheir body weight or their tumors exceeded 2000 mm³. Tumor growth in thetreated mice was compared with the average tumor growth in 37 untreatednude mice implanted with PC3 tumors.

Results

In 6 of the 12 treated mice in the study, the tumors experienced asignificant or complete growth delay (FIG. 30) relative to untreatedtumor mice, indicating that D13 was effective in slowing PC3 tumorgrowth under the conditions employed

Example 21 Cell Based Assay for Binding of KDR/VEGF Complex Binders

In this experiment the ability of a KDR/VEGF complex-binding peptide toselectively bind to the KDR/VEGF complex is demonstrated.

Reagent Preparation

The reagents for this assay were prepared as described in Example 5except where noted.

Preparation of Peptide-¹²⁵I-Streptavidin Complex Solution

Biotinylated peptides P30-XB, P31-XB, P32-XB and biotinylatednon-binding control peptide were used to prepare 1.25 μM stock solutionsin 50% DMSO. A 33.33 nM stock solution of ¹²⁵I-streptavidin waspurchased from Amersham. A stock solution of 13.33 nM 1-125streptavidin/100 nM VEGF was prepared by mixing 850 ml of 1-125streptavidin with 22 μl of 10 μM VEGF and 1275 μl of M199 media. Anotherstock solution was prepared in the same manner, but lacking VEGF. Toprepare 13.33 nM peptide-¹²⁵I-streptavidin complex solution±VEGF, 500 μlof the ¹²⁵I-streptavidin (with & without VEGF) stock solutions (preparedin last step) were mixed with 24 μl of 1.25 μM peptide solution ofP30-XB, P31-XB, P32-XB, or control peptide. The mixtures were incubatedon a rotator at 4° C. for 60 minutes, followed by addition of 50 μl ofsoft release avidin-sepharose (50% slurry in ddH₂0) to remove excesspeptides and another incubation for 30 minutes on a rotator at 4° C.Finally, the soft release avidin-sepharose was pelleted by centrifugingat 12,000 rpm for 5 minutes at room temperature, and the resultingsupernatants were used for the assays.

TABLE 9  Biotinylated Peptides SEQ Reference ID NumberStructure or Sequence NO: P30 AGPGPCKGYMPHQCWYMGTGGGK 31 P30-XBAc-AGPGPCKGYMPHQCWYMGTGGGK (Biotin-JJ)-NH₂ P31 AGMPWCVEKDHWDCWWWGTGGGK32 P31-XB Ac-AGMPWCVEKDHWDCWWWGTGGGK (Biotin-JJ)-NH₂ P32AGYGPCKNMPPWMCWHEGTGGGK 33 P32-XB Ac-AGYGPCKNMPPWMCWHEGTGGGK(Biotin-JJ)-NH₂

Binding of Peptide/Neutravidin HRP to KDR-Transfected Cells

In this assay, complexes of control peptide and the test peptides(P30-XB, P31-XB, P32-XB) with ¹²⁵I-streptavidin in the presence orabsence of VEGF (prepared as above) were tested for their ability tobind 293H cells that were transiently-transfected with KDR. The complexof P30-XB with ¹²⁵I-streptavidin specifically bound to KDR-transfected293H cells as compared to mock transfected cells in the presence of VEGF(FIG. 31A), but not when VEGF was omitted (FIG. 31B). P30-XB was alsothe best KDR/VEGF complex binder among the peptides tested usingfluorescence polarization and SPR (BiaCore) assays. See Table 9, U.S.Ser. No. 60/360,851, U.S. Ser. No. 60/440,441, and copending U.S. Ser.No. ______ entitled “KDR and VEGF/KDR Binding Peptides and Their Use inDiagnosis and Therapy,” filed on the same date as the instantapplication and incorporated by reference herein in its entirety. Thisexample shows that peptide (P30-XB) can specifically bind to TheKDR/VEGF complex present on the cell surface. Therefore, it may possiblybe used in targeting the KDR/VEGF complex in vitro and in vivo fordiagnostic or therapeutic purposes. Since the KDR/VEGF binding peptideonly detects the functional and active KDR receptor and not all the KDRpresent on cell surface, it may be useful in detecting and/or treatingactive angiogenesis in tumors, metastasis, diabetic retinopathy,psoriasis, and arthropathies. Furthermore, these peptides, as well asother peptides which bind KDR/VEGF complex may advantageously beincluded in hetermultimers of the invention.

Example 22

The following experiment assessed the ability of heterodimers D24 andD26 to block the VEGF-induced migration of HUVECs in culture anddemonstrated that the added glycosylation and/or distinct spacerstructure used in D26 enhanced its potency.

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, with or without VEGF (10 ng/mL) in the presence or absenceof D24 or D26, was added to the lower chamber of the wells. After 22hours, quantitation of cell migration/invasion was achieved bypost-labeling cells in the insert plates with a fluorescent dye andmeasuring the fluorescence of the invading/migrating cells in afluorescent plate reader. The VEGF-induced migration was calculated foreach experimental condition by subtracting the amount of migration thatoccurred when only basal medium was added to the lower chamber of thewells.

Results:

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by both D24 and D26 (FIG. 32). D26 wasten-fold more potent than D24 (IC₅₀ 0.5 nM and 5 nM respectively),indicating that the glycosylation of D26 and/or its distinct spacerproperties has enhanced its ability to bind KDR and block the effects ofVEGF.

Example 23

The following experiment assessed the ability of TK-1 (structureprovided below), a multimeric construct of the peptide TKPPR (whichbinds to NP-1, a VEGF receptor which enhances the effects of VEGFmediated by KDR), to enhance the inhibition of the VEGF-inducedmigration of HUVECs in culture produced by D6.

5CF-Gly-N{[CH₂CH₂C(═O)-Gly-N(CH₂CH₂C(═O)-Adoa-Thr-Lys-Pro-Pro-Arg-OH]₂}₂where Adoa=3,6-dioxa-8-aminooctanoyl, 5CF=5-carboxyfluoresceinyl

TK-1

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, with or without VEGF (10 ng/mL) in the presence or absenceof varying concentrations of D6, or varying concentrations of D6 incombination with a constant 100 nM TK-1 (synthesized as described in WO01/91805 A2) was added to the lower chamber of the wells. After 22hours, quantitation of cell migration/invasion was achieved bypost-labeling cells in the insert plates with a fluorescent dye andmeasuring the fluorescence of the invading/migrating cells in afluorescent plate reader. VEGF-induced migration was calculated for eachexperimental conditions by subtracting the amount of migration observedin the absence of VEGF.

Results:

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by D6 (IC₅₀ about 12.5 nM), but not by100 nM TK-1 alone (FIG. 33). Surprisingly however, TK-1 was able toenhance the potency of D6 by about ten-fold when used in the assaysimultaneously with D6 (IC₅₀ about 2.5 nM). This indicates thatcompounds containing the TKPPR sequence (or analogs) found in TK-1 canbe used to enhance the potency of certain compounds such as D6 whichcompete with VEGF for binding to KDR. In addition, a heteromultimercontaining the peptide sequences found in D6 (or similar) as well as theTKPPR sequence (or analogs), in one or more repetitions, would likelypossess enhanced activity in this assay. (See U.S. Ser. No. 09/871,974for details on the preparation of TKPPR constructs, which is hereinincorporated by reference).

Example 24 Identification of Fragments of P13-XB with KDR BindingActivity

The following experiment showed that fragments of P13-XB can maintainsignificant KDR binding activity.

Protocol:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described in Example 6. Streptavidin-HRP complexescontaining P12-XB were prepared as in Example 6. Binding of thestreptavidin-HRP complexes to the cells was carried out as in Example 6with a complex concentration of 5.5 nM in the presence of 0 to 250 nM or0 to 1000 nM of the following competing peptides: P13-XB, F1, F2, andF3. After determining the specific binding under each experimentalcondition, the IC₅₀ for each peptide was determined (where possible).

Results:

As shown in Table 9, F1, composed of just the Asp-Trp-Tyr-Tyr bindingmotif that is also shared with P12-XB along with the non-targetedGly-Gly-Gly-Lys sequence that was added to most monomeric peptidessynthesized based on phage display data, was the smallest fragment ableto block P12-XB streptavidin-HRP complex binding with an IC₅₀ below onemicromolar. Surprisingly, a larger fragment derived from P13-XB, F2,failed to significantly inhibit complex binding at one micromolar.However, when a solubilising motif, (Gly-Arg-Gly)₃ was added to thelatter peptide to make F3, it was able to compete with the complex forbinding with an IC₅₀ of 175 nM, confirming that certain fragments ofP13-XB containing the Asp-Trp-Tyr-Tyr motif retain KDR-binding activity.These fragments (or other fragments of the binding polypeptidesdisclosed herein), which retain the ability to bind the target, may beutilized instead of the full-length peptide in heteromultimers of theinvention.

TABLE 9 Fragments of P13-XB in a displacement assay competing with a complex composed of  P12-XB and streptavidin-HRP for binding to KDR-expressing cells. SEQ RefIC50, ID Number Sequence/Structure nM NO: P13-XBAc-AQDWYYDEILSMADQLRHAFLSGG-GGGK- 93 — (Biotin-JJ-)-NH₂ F1Ac-DWYYGGGK-NH₂ 850 31 F2 Ac-AQDWYYDEIL-NH₂ >1000 32 F3Ac-AQDWYYDEILJGRGRGGRGG-NH₂ 175 33

Example 25 Heterodimers Targeting Two Epitopes on a Single TargetMolecule Results in Superior Binding to a Homodimers that Binds One ofthe Two Epitopes on the Target Molecule

The following experiment provides further evidence that heterodimericconstructs are superior to homodimers in their ability to block thebiological effects of a peptide growth factor or cytokine.

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, containing either nothing or VEGF in the presence orabsence of increasing concentrations of homodimeric D8 or heterodimericD17, was added to the lower chamber of the wells. After 22 hours,quantitation of cell migration/invasion was achieved by post-labelingcells in the insert plates with a fluorescent dye and measuring thefluorescence of the invading/migrating cells in a fluorescent platereader.

Results:

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by D17 but not D8 (FIG. 34). D17blocked VEGF-induced migration with an IC₅₀ of about 250 nM while D8 hadno significant effect on migration even at 800 nM. This is in spite ofthe fact that D8 used the full targeting sequence found inp13-XB whileD17 contained a truncated version of the P13-XB sequence (as seen in F3)with a lower affinity for KDR (as demonstrated in Example 24). Thus aheterodimer with the capability of binding to two separate epitopes onthe same target molecule can be more effective at blocking ligandbinding to the target molecule than a homodimer containing the same oreven more potent targeting sequences.

Example 26 Preparation of Cyclic Peptides in which the Disulfide Bond isReplaced by an Amide Bond

Disulfide bond substitution analogs of P12-G (P12 with non-target GGGKsequence) where the Cys residues at position 6 and 13 are replaced by apair of amino acids, one with a carboxy-bearing side-chain (either Gluor Asp) and the other with an amino-bearing side chain [(Lys or Dpr(2,3-diaminopropanoic acid)] were prepared. The cycle, encompassing thesame sequence positions as those included in p12-G (made by formation ofthe disulfide bond) was made by condensation of the side-chain amino andside-chain acid moieties, resulting in a lactam ring which bridges theresidues 6-13 as does the disulfide bond of P12-G.

TABLE 10  Examples of the substitutions made for Cys⁶ and Cys¹³ of P 12-G in lactam analogs. Lactam Analogs of P12 SEQ DifferenceID Position Position in Ring Ref No. Sequence or Structure NO 6 13Size vs P12 P12-G AGPTWCEDDWYYCWLFGTGGGK 29 Cys Cys — (parent seq) P33AGPTWEEDDWYYKWLFGTGGGK 34 Glu Lys — P33-L Ac-AGPTWEEDDWYYKWLFGTGGGK-NH₂Glu Lys 4 (6-13 lactam) P34 AGPTWKEDDWYYEWLFGTGGGK 35 Lys Glu — P34-LAc-AGPTWKEDDWYYEWLFGTGGGK-NH₂ Lys Glu 4 (6-13 lactam) P35AGPTW-Dpr-EDDWYYDWLFGTGGGK-NH₂ 36 Dpr Asp — P35-LAc-AGPTW-Dpr-EDDWYYDWLFGTGGGK- Dpr Asp 0 NH₂ (6-13 lactam) P36AGPTWDEDDWYY-Dpr-WLFGTGGGK 37 Asp Dpr — P36-LAc-AGPTWDEDDWYY-Dpr-WLFGTGGGK- Asp Dpr 0 NH₂ (6-13 lactam) P37AGPTWDEDDWYYKWLFGTGGGK 38 Asp Lys — P37-L Ac-AGPTWDEDDWYYKWLFGTGGGK-NH₂Asp Lys 3 (6-13 lactam)

Representative Synthesis of Cyclic Lactam Peptides—P33-L

Synthesis of Resin Bound Peptide 1

Synthesis of 1 was carried out using Method 5 on a 0.25 mmol scale. Thepeptide resin 1 was washed and dried for further derivatizationmanually.

Synthesis of 4 P33-L

To 1 (240 mg, 0.06 mmol) was added NMM (N-methyl morpholine)/HOAc/DMF1/2/10 (v/v/v) (65 mL). Palladium tris-triphenylphosphine [Pd(PPh₃)₄,554.4 mg, 0.48 mmol] was added and the resin was shaken for 20 hshielded from light. The resin was filtered and washed with a solutionof sodium diethyldithiocarbamate (0.5 g)/DIEA (0.5 ml)/DMF (100 mL), andfinally with DMF (3×70 mL). This treatment served to expose only thecarboxy and amino groups of G1u6 and Lys13 which are required for thelactam forming reaction. The on-resin cyclization of 2 was carried outusing HATU (114 mg, 0.3 mmol), NMM (66 μL, 0.6 mmol) and DMF (10 mL) for3 h. The completion of the cyclization was monitored by Kaiser test. Thepeptide was cleaved from the peptide resin 3 using reagent B for 4 h.The resin was filtered and the filtrate was evaporated to a paste. Thecrude peptide was precipitated in ether and washed twice with ether. Thecyclic peptide was purified by preparative reverse phase linear gradientHPLC using a Waters-YMC C-18 column (250 mm×30 mm i.d.) with CH₃CN intoH₂O (both with 0.1% TFA) as the eluent. Lyophilization of theproduct-containing fractions afforded 8 mg of (P33-L). P34-L, P35-L,P36-L and P37-L were prepared similarly.

Replacement of the Disulfide Bridge of P12-G While Retaining KDR-BindingActivity

The following experiment demonstrated that the lactam P34-L, whichreplaced the chemically reactive disulfide bridge of P12-G maintainedsignificant KDR binding activity.

Protocol:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described in Example 6. Streptavidin-HRP complexescontaining P12-XB were prepared as in Example 6. Binding of thestreptavidin-HRP complexes to the cells was carried out as in Example 6with a complex concentration of 5.5 nM in the presence of 0 to 250 nMP12-G, or P34-L. After determining the specific binding under eachexperimental condition, the IC₅₀ for each peptide was determined.

Results:

As shown in Table 11, P34-L, containing a lactam disulfide bridgereplacement, was still able to compete with P12-XB-streptavidin-HRPcomplexes for binding to KDR although some affinity was lost (IC₅₀ 108nM versus 13 nM for P12-G). These lactam peptides (or similarly preparedlactam analogs of binding polypeptides disclosed herein) may be utilizedinstead of the disulfide bridge-containing peptides in heteromultimersof the invention.

TABLE 11 P12-G and P34-L (disulfide bridge replacement analog) in adisplacement assay competing with a complex composed of P12-XB andstreptavidin- HRP for binding to KDR-expressing cells. Fragment (RefNumber) IC₅₀, nM P12-G 13 P34-L 108

Example 27 Measurement of Binding of Peptide Dimers to cMet

Using a BIAcore machine, the binding constant was determined for thedimer D28 binding to immobilized cMet-Fc.

Procedure

Three densities of cMet-Fc (R&D Systems) were cross-linked to thedextran surface of a CM5 sensor chip by the standard amine couplingprocedure (3 μM solution diluted 1:100, 1:50, or 1:20 with 50 mMacetate, pH 5.5). Flow cell 1 was activated and then blocked to serve asa reference subtraction.

Final immobilization levels achieved:R_(L) Fc 2 cMet-Fc=2582R_(L) Fc 3 cMet-Fc=5048R_(L) Fc 4 cMet-Fc=9721

Experiments were performed in PBST buffer (5.5 mM phosphate, pH 7.65,0.15 M NaCl)+0.05% (v/v) Tween-20). Peptide dimers were dissolved indeionized H₂O to 1 mg/mL solutions. Dimers were diluted to 50 nM in PBS.Serial dilutions were performed to produce 25, 12.5, 6.25, and 3.125 nMsolutions. All samples were injected in duplicate. For association,dimers were injected at 30 μL/minute for 3 minutes using the kinjectprogram. Following a 10-minute dissociation, any remaining peptide wasstripped from the cMet surface with two quickinjects of 4M MgCl₂ for 2minutes at 50 μL/minute. Sensorgrams were analyzed using BIAevaluationsoftware 3.1.

Kd value of 0.79 nM was obtained for D28 (heterodimer of P26-A andP27-X), which was significantly better than KD value of eitherheterodimer alone (see SEQ ID NO:369 (880 nM) and SEQ ID NO:370 (220 nM)as shown in the Table 8 of U.S. Ser. No. ______, entitled “Peptides thatspecifically bind HGF receptor (cMet) and uses thereof,” filed on thesame date as the instant application and incorporated by referenceherein in its entirety.

Example 28 In Vitro Cell Proliferation Assay

Microvascular endothelial cells (MVECs, Cascade Biologics, Portland,Oreg.) were used to assess the in vitro efficacy of D6 and relatedanalogues for their ability to inhibit VEGF-stimulated proliferation.MVECs (passage 2) were grown to 90% confluency, trypsinized and platedin gelatin-coated 96-well microtiter plates at a density of 4-8×10³cells/well. Sixteen to 24 hours after plating, the cells were washed onetime (200 μL/well) with media devoid of fetal bovine serum butcontaining 0.1% bovine serum albumin (BSA). Fresh BSA-containing mediawas added to each well and the cells were incubated for an additional 24hours. After this 24 hour period of starvation, fresh BSA-containingmedia with or without D6 or other test substances was added and thecells were incubated for an additional 48 hours at 37° C. The media wasremoved and fresh BSA-containing media was added with or without BrdUand the cells were incubated for an additional 24 hours prior todetermining the level of incorporation exactly as described by themanufacturer (Oncogene Cat# QIA58). Results are shown in FIG. 35.

Example 29 In Vivo Inhibition of Tumor Growth

Conditions are described providing methods for determining efficacy ofthree (3) concentrations for a test compound (dimer D6) suspected ofhaving anti-angiogenic activity on SW-480 human colon carcinoma cellsusing an in vivo xenograft tumor model.

Athymic nude mice are acceptable hosts for the growth of allogenic andheterogenic cells. Nude mice are required in Points to Consider in theCharacterization of Cell Lines used to Produce Biologicals (Points toConsider in the Characterization of Cell Lines used to ProduceBiologicals, FDA 1993).

D6 is a synthetic heterodimeric peptide suspected of havinganti-angiogenic activity. This peptide binds to the human VEGF receptor2 (KDR) with high affinity and competes with VEGF binding. The followingexperiments confirms its anti-angiogenic activity.

SW-480 Human Carcinoma Cells

Colon carcinoma, SW-480, cells (ATCC) were cultured in Dulbecco'sModified Eagles Medium (DMEM) supplemented with 4 mM L-glutamine, 0.1 mMnon-essential amino acids, 50 mg/mL Gentamicin, 250 mg/mL Fungizone and10% heat inactivated fetal bovine serum at 37° C. in 95% air and 5% CO₂.

Exponentially growing cells were harvested, washed twice in phosphatebuffered saline (PBS) to remove any traces of trypsin or serum. Cellswere suspended in Hanks Balanced Salt Solution (HBSS) for injections.

Sterile phosphate buffered saline (BioWhittaker) was manufactured inaccordance with cGMP regulations and was cell culture tested to assurecompatibility; having a pH of 7.3-7.7 and an osmolarity of 271-287mOsm/kg. PBS was the vehicle used to reconstitute Test Articles and forvehicle control injections.

Cisplatin (American Pharmaceutical Partners, Inc.; Los Angeles, Calif.)was prepared according to manufacture's specifications. Cisplatin wasprepared in an aseptic fashion using a BL2 BioChem guard hood.

Test System

Species/Strain: Mus musculus, Crl:NU/NU-nuBR mice (nude mice)

Sex: Female

Age: 6-8 weeks at initiation of treatmentWeight Range No weight requirementSource: Animals were received from the Gnottobiotic Department atCharles River Laboratories, Wilmington, Mass.Number: A total of 115 animals were received and injected for thisstudy, with 90 mice used on study.

Method of Identification:

Mice were uniquely numbered using an ear tag system. Additionally, cageswere marked with cage cards minimally identifying group number, animalnumber, study number and IACUC protocol number.

Randomization:

Animals were randomly assigned to treatment groups using Microsoft®Excel 97 SR-1 program.

Animal Care

Mice were fed gamma-irradiated rodent chow ad libitum. Tap water wassterilized and supplied via bottle and sipper tube ad libitum.

Animal Environment:

Animals were housed by groups in semi-rigid isolators. Mice were housedin flat bottom caging containing five to ten animals. Cages containedgamma-irradiated contact bedding. The number of mice in each cage mayhave been altered due to the behavior of the mice, changes were noted inthe isolator inventory. The housing conforms to the recommendations setforth in the Guide for the Care and Use of Laboratory Animals, NationalAcademy Press, Washington, D.C., 1996 and all subsequent revisions.

Environmental controls were set to maintain a temperature of 16-26° C.(70±8° F.) with a relative humidity of 30-70. A 12:12 hour light: darkcycle was maintained.

Acclimation:

Once animals were received, they were allowed to acclimate to thelaboratory environment for 24-hours prior to the study start. Mice wereobserved for signs of disease, unusual food and/or water consumption orother general signs of poor condition. At the time of animal receipt,animals were clinically observed and appeared to be healthy.

Experimental Design:

Female athymic nude mice (Crl:NU/NU-nuBR) at 6-8 weeks of age were usedin this study. A total of 115 mice were injected subcutaneously into theright lateral thorax with 5×10⁶ SW-480, human colon carcinoma cells.When tumors reached a target window size of approximately 150±75 mg, 90tumor-bearing mice were randomly selected and distributed into one ofnine groups. Test compound and vehicle were administeredintraperitoneally (IP), Cisplatin was administered intravenously (IV).Tumor measurements were recorded twice weekly using hand-held calipers.Mice were monitored daily for signs of toxicity and morbidity. At studytermination, animals were euthanized by carbon dioxide overdose andnecropsied for tissue collection.

A total of nine (9) groups were used in this study. Each group containedten (10) tumor-bearing mice. Groups 1 and 2 contained untreated andvehicle treated negative control mice, respectively. Groups 3, 4, and 5contained mice that received one of three different concentrations ofthe D6 heterodimer. Groups 6, 7, and 8 contained mice that received oneof three different concentrations of a different anti-angiogenicpeptide. Group 9 contained mice that received cisplatin, a standardchemotherapeutic compound as a positive control.

Dose levels for each group are provided in Table 12. Dosing began thesame day that animals were randomly sorted into groups (Study Day 7).Each dose was removed from the dose vial using aseptic technique foreach animal and the injection site was wiped with an alcohol swab priorto dose administration. Doses were administered with a 1.0 mL syringeand a 27-gauge×½″ needle for each mouse.

TABLE 12 Study Treatment Groups Group Test Compound Concentration mg/kgNumber of Animals 1 Untreated — 10 2 Vehicle 0 10 3 D6 0.05 10 4 D6 0.510 5 D6 5.0 10 9 Cisplatin 6.0 10

The Test compound- and vehicle-treated mice received dailyintraperitoneal (IP) injections for 15 days. Cisplatin was administeredevery other workday for a total of five (5) doses via an intravenousroute.

Clinical Observations of each animal were performed and recorded atleast once daily for toxicity, morbidity and mortality. Morbidityincluded signs of illness such as, but not limited to, emaciation,dehydration, lethargy, hunched posture, unkempt appearance, dyspnea andurine or fecal staining

Tumor Measurements:

In accordance with the protocol, tumor measurements were taken twiceweekly throughout the study by measuring the length and width of tumorswith calibrated calipers. Measurements occurred a minimum of 3-4 daysapart, except when animals were euthanized and measurements were taken;this sometimes resulted in an interval of less than 3 days. Tumorweights were calculated using the following formula: mg=(L×W²)/2.Animals were euthanized either when mean tumor weight was >1000 mg pergroup over two (2) consecutive measurements, or if tumors becameulcerated, impaired the animal's ability to ambulate or obtain food andwater.

Unscheduled Euthanasia and Unexpected Deaths: 1. Unscheduled Euthanasia:

None of the animals required unscheduled euthanasia while on study.

2. Unexpected Deaths:

None of the animals died while on study.

Necropsy: 1. Euthanasia and Necropsy Order:

All mice in groups 1, 2, 3, 4, and 5 (50 total) were submitted fornecropsy when tumors reached a group mean target size of ≧1000 mg overtwo (2) consecutive measurements within a group. Animals were submittedfor necropsy to the Charles River Laboratories Health MonitoringLaboratory (HM), Wilmington, Mass. All animals were euthanized on StudyDay 22, short of received the full 28 day treatment regiment with TestArticles because mean tumor size was ≧1000 mg in Test Article TreatedGroups 3-8. All animals were humanely euthanized by carbon dioxide (CO₂)inhalation.

Tissue Collection:

Tumors were dissected free of surrounding tissue and overlying skin.Additionally the kidneys were collected. Any abnormalities noted on therenal surfaces were noted.

Frozen blocks were made of tumors and kidneys for each animal. Arepresentative section of the tissue (tumor, kidneys) was taken. Kidneysections included the cortex and medulla. Tissue sections were placed inthe bottom of a labeled plastic-freezing mold. Tissue was embedded withOCT medium. Blocks were submerged into isopentane chilled with dry iceuntil frozen. Blocks were briefly examined for quality, and stored ondry ice.

Blocks were labeled with the animal number and a letter codecorresponding to tissue (A=left kidney; B=right kidney; C=mass). Blocksfrom one animal were placed into a labeled bag.

Results: A. In-Life Measurements and Observations: 1. ClinicalObservations, Morbidity and Mortality Summary Statement:

All animals appeared healthy and were within normal limits throughoutthe study and the Test Compound (D6) did not show any signs of toxicityat the doses used in this study.

Animals were euthanized on Study Day 22. All mice, except Group 9 mice,were euthanized prior to completing Test compound administration,because mean tumor size was ≧1000 mg in Groups 1-8. Group 9,Cisplatin-treated animals were euthanized on Study Day 22 when meantumor weight was 995 mg. No animals died while on study.

Mass Palpation Summary

Throughout the study palpable masses were detected in all mice, withtumors progressively growing for the duration of the study. As expectedtumors in untreated and vehicle treated negative control mice (Groups 1and 2) grew the fastest, reaching a mean tumor size of 1000 mg on orbefore Study Day 20. In addition, animals treated with Cisplatin (Group9) developed tumors that grew the slowest reaching a mean tumor size of995 mg at study termination (Day 22).

In general, except for Group 3 mice, all animals treated with Testcompounds resulted in slower tumor growth. Animals in Group 3, whichwere treated with the low dose of D6 (0.05 mg/kg) had tumors that grewat approximately the same rate as the tumors in untreated and vehicletreated animals in Groups 1 and 2. Animals treated with higher doses ofD6 (Groups 4 and 5) had tumors that grew slower; reaching a mean tumorsize of 1000 mg on Study Day 21. When compared to control Groups 1 and 2mice, Test compound treatment resulted in a delay of tumor growth ofapproximately 1 day.

CONCLUSIONS

Data from this study validate the model used because tumor-bearing micein negative control Groups 1 and 2 and positive control Group 9responded as expected.

Throughout the study palpable masses were observed in all groups. Inaddition, all animals were healthy and within normal limits throughoutthe study. Furthermore, the Test compound (D6) did not appear toadversely affect the animals. Therefore, these data would suggest thatanimals treated with D6 had tumors that grew slowly (approximately 1 dayslower over the 22 day test period than controls). Also, since the Testcompound did not show any significant toxic effects, higherconcentrations of Test compound could also be used with potentiallybetter tumor regression.

Example 30

The following example describes the preparation of an ultrasoundcontrast agent conjugated to a KDR-binding heterodimer of the inventionand the ability of the heterdimer conjugated contrast agent to localizeto KDR-expressing cells in vitro and angiogenic tissue in vivo.

Preparation of Derivatized Microbubbles for Peptide Conjugation

200 mg of DSPC (distearoylphosphatidylcholine), 275 mg of DPPG.Na(distearoylphosphatidylglycerol sodium salt) and 25 mg of N-MPB-PE weresolubilized at 60° C. in 50 ml of Hexan/isopropanol (42/8). The solventwas evaporated under vacuum, and then PEG-4000 (35.046 g) was added tothe lipids and the mixture was solubilized in 106.92 g of t-butylalcohol at 60° C., in a water bath. The solution was filled in vialswith 1.5 ml of solution. The samples were rapidly frozen at −45° C. andlyophilized. The air in the headspace was replaced with a mixture ofC₄F₁₀/Air (50/50) and vials capped and crimped. The lyophilized sampleswere reconstituted with 10 ml saline solution (0.9%-NaCl) per vial,yielding a suspension of phospholipids stabilized microbubbles.

Peptide Conjugation

D23 (a dimeric construct of P6- and P12-derived sequences) wasconjugated with a preparation of microbubbles as above described,according to the following methodology. The thioacetylated peptide (200μg) was dissolved in 20 μl DMSO and then diluted in 1 ml of PhosphateBuffer Saline (PBS). This solution was mixed to the N-MPB-functionalizedmicrobubbles dispersed in 18 ml of PBS-EDTA 10 mM, pH 7.5 and 2 ml ofdeacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 Mhydroxylamine.HCl, pH 7.5) was added. The headspace was filled withC₄F₁₀/Air (50/50) and the mixture was incubated for 2.5 hours at roomtemperature under gentle agitation (rotating wheel), in the dark.Conjugated bubbles were washed by centrifugation. Similarly, the monomerpeptides making up D23 were separately conjugated to two differentmicrobubble preparations according to the methodology described above.

In Vitro Assay on Transfected Cells

The ability of phospholipid stabilized microbubbles conjugated toheteromultimeric constructs of the invention to bind to KDR-expressingcells was assessed using 293H cells transfected to express KDR.

Transfection of 293H Cells on Thermanox® Coverslips:

293H cells were transfected with KDR DNA as set forth in Example 6. Thetransfected cells were incubated with a suspension of peptide-conjugatedmicrobubbles prepared as described above. For the incubation with thetransfected cells a small plastic cap is filled with a suspensioncontaining 1 to 3.10⁸ peptide-conjugated microbubbles and the capcovered with an inverted Thermanox® coverslip is placed so that thetransfected cells are in contact with the conjugated microbubbles. Afterabout 20 min at RT, the coverslip is lifted with tweezers, rinsed threetimes in PBS and examined under a microscope to assess binding of theconjugated microbubbles.

Determination of the % of Surface Covered by Microvesicles

Images were acquired with a digital camera DC300F (Leica) and thepercent of surface covered by bound microbubbles in the imaged area wasdetermined using the software QWin (Leica Microsystem AG, Basel,Switzerland). Table 13 shows the results of the binding affinity(expressed as coverage % of the imaged surface) of targetedmicrovesicles of the invention to KDR transfected cells, as compared tothe binding of the same targeted microvesicles to Mock-transfectedcells.

TABLE 13 Conjugated microbubbles prepared as described above % ofcovered surface Peptide code Batch Id KDR Mock P6 Derivative BG1979T023.5% 0.9% P12 Derivative BG1980T02 16.8% 1.0% D23 (dimer) BG2002T0222.9% 3.3% D6 Deriv./ BG1958T02 12.9% 0.8% P12 Deriv.

When the P-6 derived sequence and the P12-derived sequence areseparately attached to phospholipid stabilized microbubbles as monomersthe resulting preparations achieve binding of the bubbles to KDRtransfected cells in vitro to a different extent (3.5% & 16.8%). When apreparation of phospholipid stabilized microbubbles resulting from theaddition of equal quantities of each of these peptide monomers (but thesame total peptide load) is tested in the same system 12.9% binding isachieved. Binding is a little more than the average of the two but as itis achieved with two sequences that bind to different sites on thetarget will be more resistant to competition at one or other of thesites on the target. However, for D23, the dimer, binding is increasedto 22.9% (with the same peptide load). These results indicate thathetromultimers of the invention permit increased binding and increasedresistance to competition.

In Vivo Animal Models

A known model of angiogenic tissue (the rat Mat B III model) was used toexamine the ability of phospholipid stabilized microbubbles conjugatedto a heteromultimer of the invention to localize to and provide imagesof angiogenic tissue.

Animals: Female Fisher 344 rat (Charles River Laboratories, France)weighing 120 to 160 g were used for the MATBIII tumor implantation. MaleOFA rats (Charles River Laboratories, France) weighing 100 to 150 g wereused for Matrigel injection.

Anesthesia

Rats were anesthetized with an intramuscular injection (1 ml/kg) ofKetaminol®/xylazine (Veterinaria AG/Sigma) (50/10 mg/ml) mixture beforeimplantation of Matrigel or MatBIII cells. For imaging experiments,animals were anesthetized with the same mixture, plus subcutaneousinjection of 50% urethane (1 g/kg).

Rat MATBIII Tumor Model

A rat mammary adenocarcinoma, designated 13762 Mat B III, was obtainedfrom ATCC(CRL-1666) and grown in McCoy's 5a medium+10% FCS. 1% glutamineand 1% pen/strep (Invitrogen cat# 15290-018). Cells in suspension werecollected and washed in growth medium, counted, centrifuged andresuspended in PBS or growth medium at 1.10⁷ cells per ml. For tumorinduction: 1×10⁶ cells in 0.1 ml were injected into the mammary fat padof anesthetized female Fisher 344 rat. Tumors usually grow to a diameterof 5-8 mm within 8 days.

In Vivo Ultrasound Imaging

Tumor imaging was performed using an ultrasound imaging system ATL HDI5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversionat low acoustic power (MI=0.05) was used to follow accumulation ofpeptide conjugated-microbubbles on the KDR receptor expressed on theendothelium of neovessels. For the control experiments, an intravenousbolus of unconjugated microbubbles or microbubbles conjugated tonon-specific peptide was injected. The linear probe was fixed on theskin directly on line with the implanted tumors and accumulation oftargeted bubbles was followed during thirty minutes.

A perfusion of SonoVue® was administrated before injecting the testbubble suspension. This allows to evaluate the vascularization statusand the video intensity obtained after SonoVue® injection is taken as aninternal reference.

A baseline frame was recorded and then insonation was stopped during theinjection of the microbubbles. At various time points after injection(1, 2, 5, 10, 15, 20, 25, 30 minutes) insonation was reactivated and 2frames of one second were recorded on a videotape.

Video frames from tumor imaging experiments were captured and analysedwith the video-capture and Image-Pro Plus 2.0 software respectively. Thesame rectangular Area of Interest (AOI) including the whole sectionalarea of the tumor was selected on images at different time points (1, 2,5, 10, 15, 20, 25, 30 minutes). At each time point, the sum of the videopixel inside the AOI was calculated after the subtraction of the AOIbaseline. Results are expressed as the percentage of the signal obtainedwith SonoVue®, which is taken as 100%. Similarly, a second AOI situatedoutside the tumor, and representing the freely circulating contrastagent, is also analyzed.

FIG. 37 shows uptake and retention of bubble contrast in the tumor up to30 minutes post injection for suspensions of phospholipid stabilizedmicrobubbles conjugated to a heteromultimeric construct of the inventionprepared as described above (D23). In contrast, the same bubbles showedonly transient (no more than 10 minutes) visualization/bubble contrastin the AOI situated outside the tumor site.

Other Embodiments

Although the present invention has been described with reference topreferred embodiments, one skilled in the art can easily ascertain itsessential characteristics and without departing from the spirit andscope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Those skilled inthe art will recognize or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are encompassed in thescope of the present invention.

All publications and patents mentioned in this specification are hereinincorporated by reference.

1. A multivalent compound comprising at least two binding moietieshaving specificity for different binding sites on the same target. 2.The compound of claim 1, wherein said compound is a multimeric compoundcomprising a plurality of binding moieties.
 3. The compound of claim 1,wherein said compound is a dimeric compound.
 4. The compound of claim 1,wherein at least one of the binding moieties comprises a polypeptide. 5.The compound of claim 4, wherein all of the binding moieties comprisepolypeptides.
 6. The compound of claim 5, wherein the affinity of thecompound for the target is about 60 fold greater than the affinity ofany one of the polypeptides for the target.
 7. The compound of claim 5,wherein the affinity of the compound for the target is about 560 foldgreater than the affinity of any one of the polypeptides for the target.8. The compound of claim 4 or 5, wherein each polypeptide is selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12.
 9. The compound of claim 4 or 5,wherein each polypeptide is selected from the group consisting of SEQ IDNO: 26, SEQ ID NO: 27, SEQ NO: 28, and SEQ ID NO: 29
 10. The compound ofclaim 8 or 9, wherein the polypeptide comprises an amino acidsubstitution, and amide bond substitution, a D-amino acid substitution,a glycosylated amino acid, a disulfide mimetic substitution, an aminoacid translocation, a retroinverso peptide, a peptoid, a retro-inversopeptoid, or a synthetic peptide.
 11. The compound of claim 1, whereinthe target is a protein
 12. The compound of claim 1, wherein the targetis a receptor or a receptor/ligand complex.
 13. The compound of claim11, wherein the binding moieties bind to different epitopes on theprotein.
 14. The compound of claim 12, wherein the binding moieties bindto different epitopes on the receptor or receptor/ligand complex. 15.The compound of claim 11, wherein said target is a receptor involved inangiogenesis.
 16. The compound of claim 12, wherein said receptor is aprotein-tyrosine kinase receptor.
 17. The compound of claim 1, whereinthe target comprises KDR or KDR/VEGF complex.
 18. The compound of claim17, wherein the binding moieties bind to different epitopes on KDR orKDR/VEGF complex.
 19. The compound of claim 11, wherein said target is areceptor involved in hyperproliferation.
 20. The compound of claim 11,wherein said target is a receptor expressed on a tumor.
 21. The compoundof claim 1, wherein the target comprises hepatocyte growth factor (HGF)receptor (cMet) or HGF/cMet complex.
 22. The compound of claim 21,wherein the binding moieties bind to different epitopes on cMet or theHGF/cMet complex.
 23. The compound of claim 22, wherein the bindingmoieties comprise polypeptides.
 24. The compound of claim 18, whereinthe binding moieties are selected from the group consisting of SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:12.
 25. The compound ofclaim 18, wherein the binding moieties comprise SEQ ID NO:4 and SEQ IDNO:5.
 26. The compound of claim 22, wherein the binding moieties areselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQNO: 28, and SEQ ID NO:
 29. 27. The compound of claim 1, furthercomprising at least one labeling group or therapeutic agent.
 28. Thecompound of claim 27, wherein the target comprises KDR or KDR/VEGFcomplex.
 29. The compound of claim 28, wherein the binding moieties bindto different epitopes on KDR or KDR/VEGF complex.
 30. The compound ofclaim 29, wherein the target comprises the hepatocyte growth factor(HGF) receptor (cMet) or HGF/cMet complex.
 31. The compound of claim 30,wherein the binding moieties bind to different epitopes on cMet or theHGF/cMet complex.
 32. The compound of claim 27, wherein the bindingmoieties comprise the sequence SEQ ID NO:8 and SEQ ID NO:9.
 33. Thecompound of claim 27, wherein the binding moities comprise the sequenceSEQ ID NO:4 and SEQ ID NO:5.
 34. The compound of claim 27, wherein thebinding moieties are selected from the group consisting of SEQ ID NO:26, SEQ ID NO: 27, SEQ NO: 28, and SEQ ID NO:
 29. 35. The compound ofclaim 27, wherein the labelling group or therapeutic agent comprises oneor more paramagnetic metal ions or superparamagnetic particles, anultrasound contrast agent, one or more photolabels, or one or moreradionuclides.
 36. The compound of claim 35, wherein the paramagneticmetal ion is selected from Mn²⁺, Cu²⁺, Fe²⁺, Co²⁺, Ni²⁺, Gd³⁺, Eu³⁺,Dy³⁺, Pr³⁺, Cr³⁺, Co³⁺, Fe³⁺, Ti³⁺, Tb³⁺, Nd³⁺, Sm³⁺, Ho³⁺, Er³⁺, Pa⁴⁺,and Eu²⁺.
 37. The compound of claim 35, further comprising a chelator,wherein the chelator is 1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10teraazacyclododecane triacetic acid (DO3A).
 38. The compound of claim35, further comprising gadolinium (III).
 39. The compound of claim 35,wherein the ultrasound contrast agent comprises a phospholipidstabilized microbubble or a microballoon comprising a fluorinated gas.40. The compound of claim 35, wherein the labeling group or therapeuticagent further comprises a chelator.
 41. The compound of claim 40,wherein the chelator comprises DTPA, DOTA, EDTA, TETA, EHPG, HBED, NOTA,DOTMA, TETMA, PDTA, TTHA, LICAM, or MECAM.
 42. The compound of claim 40,wherein the chelator comprises diethylenetriamine pentaacetic acid,tetraazacyclododecane triacetic acid, or a carboxymethyl-substitutedderivative thereof.
 43. The compound of claim 35, where the radionuclideis ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, ⁷⁶Br, ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga,⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm,¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb,²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb,¹⁷⁷Lu, ¹⁹⁸Au or ¹⁹⁹Au.
 44. The compound of claim 43, further comprisinga compound having a structure selected from the following:


45. The compound of claim 43, further comprising a compound having astructure selected from the following:

where X is CH₂ or O; Y is C₁-C₁₀ branched or unbranched alkyl, aryl,aryloxy, arylamino, arylaminoacyl, or aralkyl comprising C₁-C₁₀ branchedor unbranched alkyl groups, C₁-C₁₀ branched or unbranched hydroxy orpolyhydroxyalkyl groups or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups; J is C(═O)—, OC(═O)—, SO₂—, NC(═O)—,NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—, a homopolyamide or aheteropolyamine derived from synthetic or naturally occurring aminoacids; and n is 1-100.
 46. The compound of claim 43, further comprisinga compound having the following structure:


47. The compound of claim 44 or 45, further comprising ^(99m)Tc, ¹⁸⁶Re,or ¹⁸⁸Re.
 48. The compound of claim 46, further comprising ^(99m)Tc. 49.The compound of claim 43, further comprising a compound having thefollowing structure:

where R is an alkyl group.
 50. The compound of claim 43, furthercomprising a compound having the following structure:

where R is an alkyl group.
 51. The compound of claim 43, furthercomprising a compound having the following structure:


52. The compound of claim 49, 50 or 51, further comprising ¹⁷⁷Lu, ⁹⁰Y,¹⁵³Sm, ¹¹¹In, or ¹⁶⁶Ho.
 53. The compound of claim 27, further comprisinga linker between a binding moiety and the labelling group or therapeuticagent.
 54. The compound of 53, wherein the linker comprises asubstituted alkyl chain, an unsubstituted alkyl chain, a polyethyleneglycol derivative, an amino acid spacer, a sugar, an aliphatic spacer,an aromatic spacer, a lipid molecule, or combination thereof.
 55. Thecompound of claim 27, wherein the therapeutic agent comprises abioactive agent, a cytotoxic agent, a drug, a chemotherapeutic agent, ora radiotherapeutic agent.
 56. The compound of claim 3, wherein saidcompound comprises a dimer selected from D1, D4, D5, D6, D7, D10, D13,D17, D24, D26, and D31.
 57. The compound of claim 3, wherein saidcompound comprises a dimer having the following formula:


58. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


59. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


60. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


61. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


62. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


63. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


64. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


65. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula:


66. A diagnostic imaging agent comprising a compound of any of claim 1,46, or 47 conjugated to a microbubble or microballoon.
 67. The imagingagent of claim 66, wherein said microbubble or microballoon comprises aphospholipid comprising the formula:


68. The imaging agent of claim 66, wherein said microbubble ormicroballoon comprises an biocompatible fluorinated gas selected fromthe group consisting of SF₆, freons, and perfluorocarbons.
 69. Adiagnostic imaging method comprising the steps of: (a) administering toa patient a pharmaceutical preparation comprising a compound accordingto any one of claim 1, 53, or 54; and (b) imaging the compound afteradministration to the patient.
 70. The method of claim 69, wherein theimaging step comprises magnetic resonance imaging, ultrasound imaging,optical imaging, sonoluminescence imaging, photoacoustic imaging, ornuclear imaging.
 71. The method of claim 69, wherein the administeringstep comprises inhaling, transdermal absorbing, intramuscular injecting,subcutaneous injecting, intravenous injecting, or intraarterialinjecting.
 72. A method of treating a disease, comprising the step ofadministering to a patient a pharmaceutical preparation comprising acompound of claim 49-55.
 73. A method of treating a disease associatedwith angiogenesis, comprising the step of administering to a patient apharmaceutical preparation comprising a compound of claim 28, 29 or32-33.
 74. A method of treating a disease associated withhyperproliferation, comprising the step of administering to a patient apharmaceutical preparation comprising a compound of claim 31, 31 or 34.75. The method of claim 73 or 74 wherein the disease is neoplastic tumorgrowth.
 76. A method of treating a disease comprising the step ofadministering to a patient a pharmaceutical preparation comprising acompound of claim
 1. 77. A method of screening for heteromultimericcompounds having improved binding affinity, the method comprising: (a)preparing a labeled compound comprising at least two binding moietiesthat bind to different binding sites of a target; (b) contacting thelabeled compound with a target; (c) determining a dissociation constantof the labeled compound; and (d) comparing the dissociation constant ofthe labeled compound with the dissociation constant of one or moreindividual binding moieties.
 78. The method of claim 77, wherein saidcompound is a heteromultimeric compound comprising a plurality ofbinding moieties.
 79. The method of claim 77, wherein said compound is aheterodimeric compound.
 80. The method of claim 77, wherein at least oneof the binding moieties comprises a polypeptide.
 81. The method of claim77, wherein the target is a protein
 82. The method of claim 77, whereinthe target is a receptor or a receptor/ligand complex
 83. The method ofclaim 77, wherein the binding moieties bind to different epitopes on theprotein.
 84. The method of claim 77, wherein the binding moieties bindto different epitopes on the receptor or the receptor/ligand complex.85. The method of claim 77, wherein said target is a receptor involvedin angiogenesis or hyperproliferation.
 86. The method of claim 85,wherein said receptor is a protein-tyrosine kinase receptor.
 87. Themethod of claim 82, wherein the target comprises KDR or VEGF/KDRcomplex.
 88. The method of claim 87, wherein the binding moieties bindto different epitopes n KDR or KDR/VEGF complex.
 89. The method of claim82, wherein the target comprises hepatocyte growth factor (HGF) receptor(cMet) or HGF/cMet complex.
 90. The method of claim 89, wherein thebinding moieties bind to different epitopes on cMet or the HGF/cMetcomplex.
 91. The method of claim 77, wherein at least one of the bindingmoieties comprises the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12.
 92. The method of claim 77,wherein the binding moieties comprise SEQ ID SEQ ID NO: 26, SEQ ID NO:27, SEQ NO: 28, or SEQ ID NO:
 29. 93. The method of claim 78, furthercomprising the step of: identifying a labeled heteromultimeric compoundhaving a dissociation constant that is about twenty-fold less than thedissociation constant of a constituent binding moiety.
 94. The compoundof claim 39, wherein the target is a receptor or a receptor/ligandcomplex.
 95. The compound of claim 94, wherein the binding moieties bindto different epitopes on the receptor or receptor/ligand complex. 96.The compound of claim 95, wherein said target is a receptor involved inangiogenesis.
 97. The compound of claim 96, wherein said receptor is aprotein-tyrosine kinase receptor.
 98. The compound of claim 97, whereinthe target comprises KDR or KDR/VEGF complex.
 99. The compound of claim98, wherein the binding moieties bind to different epitopes on KDR orKDR/VEGF complex.
 100. The compound of claim 95, wherein said target isa receptor involved in hyperproliferation.
 101. The compound of claim 96or 100, wherein said target is a receptor expressed on a tumor.
 102. Thecompound of claim 100, wherein the target comprises hepatocyte growthfactor (HGF) receptor (cMet) or HGF/cMet complex.
 103. The compound ofclaim 95, wherein the binding moieties comprise polypeptides.
 104. Thecompound of claim 103, wherein the polypeptides are selected from thegroup consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, and SEQ IDNO:12.
 105. The compound of claim 103, wherein the polypeptides areselected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5. 106.The compound of claim 103, wherein the polypeptides are selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ NO: 28, andSEQ ID NO:
 29. 107. A method of synthesizing a multimeric compoundcomprising at least two binding moieties having specificity fordifferent binding sites on the same target, wherein at least one of thebinding moieties comprises a cyclic polypeptide formed by introducing anamide bond between two side chains.
 108. A method of synthesizing amultimeric compound comprising at least two binding moieties havingspecificity for different binding sites on the same target, wherein atleast one of the binding moieties comprises a polypeptide and a linkercomprising at least one glycosylated amino acid selected from the groupconsisting or serine, threonine and homoserine.
 109. A method ofsynthesizing a multimeric compound comprising at least two bindingmoieties having specificity for different binding sites on the sametarget, selected from the group consisting of D1, D4, D5, D9, D10, D11,D12, D13, D14, D15, D16, D17, D18, D19, D₂O, D21, D22, D23, D24, D25,D26 and D27, wherein the method comprises the steps set forth in Example9.
 110. The compound of claim 3, wherein said compound comprises a dimerhaving the following formula: